Method of transforming soybean

ABSTRACT

The present disclosure provides methods for Agrobacterium-mediated transformation of soybean cells or tissue and regeneration of the transformed cells or tissue into transformed plants. The methods may be used for transforming many soybean cultivars.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. ProvisionalApplication Serial No. 60/390,562, filed Jun. 22, 2002, the entirecontents of which is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The invention relates generally to methods for planttransformation and, more particularly, to methods for transformingsoybean cells or tissues. The invention also relates to methods forregenerating transgenic soybean plants from transformed soybean cells ortissues. The invention also relates to transgenic soybean plants andseeds obtained by such methods.

BACKGROUND

[0003] Soybean is a major food and feed source that is grown on moreacres worldwide than any other dicotyledonous crop. It is reportedlygrown on more than 50 million hectares. Unfortunately, only a few plantintroductions have given rise to the major cultivars grown in the UnitedStates and, as a consequence, this narrow germplasm base has limitedsoybean breeding potential. The limited genetic base in domestic soybeanvarieties has limited the power of traditional breeding methods todevelop varieties with improved or value-added traits.

[0004] Hence, the use of genetic engineering techniques to modifysoybean can facilitate the development of new varieties with, forexample, traits such as herbicide resistance, disease resistance (suchas virus resistance, for example), and seed quality improvement in amanner that has been unattainable by traditional breeding methods ortissue-culture induced variation.

[0005] The development of an efficient transformation system isnecessary for the analysis of gene expression in plants. Therequirements for such a system include a proper target plant tissue thatwill allow efficient plant regeneration, a gene delivery vehicle thatdelivers foreign DNA efficiently into the target plant cells, and aneffective method for selecting transformed cells. In genetictransformation of dicotyledonous species, transformation systemsutilizing the bacterium Agrobacterium tumefaciens have been frequentlyused as vehicles for gene delivery. The preferred target tissues forAgrobacterium-mediated transformation presently include cotyledons, leaftissues, and hypocotyls. High velocity microprojectile bombardmentoffers an alternative method for gene delivery into dicotyledonousplants.

[0006] Agrobacterium-mediated gene delivery in soybean has been far fromroutine. In reports that have been available to the public, meristemsand cotyledon tissues have been frequently mentioned as targets for usein Agrobacterium-mediated gene delivery. However, reliable and efficienttransformation and regeneration from these two explant sources are oftennot accomplished.

[0007] U.S. Pat. No. 5,169,770 and U.S. Pat. No. 5,376,543 to Chee etal. discuss a non-tissue culture method of transforming soybeans toproduce transgenic plants, wherein seeds are germinated and meristematicor mesocotyl cell tissues are inoculated with bacterial cells,specifically Agrobacterium strains, which, through infection, transferDNA into the explants. This method depends on the growth of preformedshoots.

[0008] Parrott W. A. et al. (1989), “Recovery of primary transformantsof soybean,” Plant Cell Reports 7:615-617, report recovery of soybeantransformants from immature cotyledon tissue after co-cultivation withAgrobacterium. However, the regenerated plants were chimeric, and thetransgenes were not transmitted to the progeny.

[0009] U.S. Pat. No. 5,416,011 (to Hinchee et al.) discusses utilizing acotyledon explant, which requires removal of the hypocotyl, saving andseparating the cotyledons and inserting a chimeric gene by inoculationwith Agrobacterium tumefaciens vectors containing the desired gene.

[0010] Yan B. et al. (2000), “Agrobacterium tumefaciens-mediatedtransformation of soybean using immature zygotic cotyledon explants,”Plant Cell Reports 19:1090-1097, report an overall 0.03% transformationfrequency in Agrobacterium-mediated transformation in soybean withimmature cotyledons.

[0011] U.S. Pat. No. 6,384,301 to Martinell et al. describesAgrobacterium-mediated gene delivery into cells in the meristem of anisolated soybean embryonic axis. Their method does not involve acallus-phase tissue culture.

[0012] From the work described above, it is clear that the goal ofestablishing a reliable soybean transformation system is seldomaccomplished by the workers involved when meristems and cotyledontissues are used as source explants for Agrobacterium-mediated genedelivery. Therefore, there is a need to continue to exploit newmethodology, including new source explants, in order to develop a moreefficient soybean transformation system.

[0013] It has been demonstrated in soybean tissue culture that plantregeneration may be achieved from epicotyl tissues and primary leaftissues. However, to-date, no successful transformation has beenreported in soybean using these two explant sources as targets for genedelivery.

[0014] Wright M. S. et al. (1987) “Initiation and propagation of Glycinemax L. Merr.: Plants from tissue-cultured epicotyls,” Plant Cell Reports8:83-90, describes successful initiation and proliferation of shootsfrom epicotyl tissue of soybean. Explanted epicotyls were induced toform shoots in Schenk and Hildebrandt medium containing 20 μM kinetinfor 5 weeks. Shoot proliferation was maintained on N6 medium containing2.1 nM picloram and 0.1 μM benzyladenine.

[0015] Wright M. S. et al. (1987) “Regeneration of soybean (Glycine maxL. Merr.) from cultured primary leaf tissue,” Plant Cell Reports6:83-89, describes a reproducible method for regeneration of plants fromprimary leaf tissue of 27 varieties of soybean They found that while2,4,5-trichlorophenoxyacetic acid was demonstrated to be essential forregeneration, addition of benzyadenine (BA) was found to enhanceregeneration.

[0016] Rajasekaren K. et al. (1997) “Somatic embryogenesis from culturedepicotyls and primary leaves of soybean (Glycine max L. Merr),” In VitroCellular & Developmental Biology 33(2):88-91, describes regeneration ofseveral varieties of soybean by somatic embryogenesis from culturedepicotyls and primary leaf tissues of immature seeds from greenhousegrown plants. They found that somatic embryogenesis was induced fromepicotyls and primary leaves when cotyledon halves with the intactzygotic embryo axes were cultured on Murashige and Skoog (MS) mediumsupplemented with 46.2 μM 2,4-D. In the absence of being cultured withthe cotyledon halves, no embryogenesis was observed from isolated axes,epicotyls or primary leaves. Rapid multiplication of shoot tips fromgerminating somatic embryos was achieved on Cheng's basal mediumcontaining 11.3 μM benzyladenine.

SUMMARY

[0017] The present invention provides a method for transforming soybeancells and regeneration of the transformed cells into transformed plants.The method may be used for transforming many soybean cultivars.

[0018] The invention provides a novel soybean explant that enablesAgrobacterium tumefaciens-mediated gene delivery into soybean cells withhigh efficiency.

[0019] In particular, the invention provides a method for transformingsoybean cells or tissue, the method comprising:

[0020] (a) preparing an explant from a soybean seed by:

[0021] (i) removing all or a part of the hypocotyl from said seed;

[0022] (ii) removing one cotyledon along with its adjacent axillary budfrom the seed, and leaving one cotyledon with the epicotyl and primaryleaves attached thereto;

[0023] (iii) removing a portion of a primary leaf from the remainingcotyledon, thereby generating a primary leaf base; and

[0024] (b) co-cultivating the explant with Agrobacterium comprising anucleic acid of interest to be incorporated into the genome of thesoybean cells.

[0025] In additional embodiments, the method further includes one ormore of the following: inducing shoot formation from the primary leafbase and the adjacent epicotyl; cultivating the shoot in a mediumcontaining a selection agent; selecting a transformed shoot; andregenerating a transformed plant from the transformed shoot.

[0026] In a further embodiment, the invention provides a method forproducing a stably transformed soybean plant, the method comprising:

[0027] (a) preparing an explant from a soybean seed by:

[0028] (i) removing all or a part of the hypocotyl from said seed;

[0029] (ii) removing one cotyledon along with its adjacent axillary budfrom the seed, and leaving one cotyledon with the epicotyl and primaryleaves attached thereto;

[0030] (ii) removing a portion of a primary leaf from the epicotyl,thereby generating at least one primary leaf base;

[0031] (b) co-cultivating the explant with Agrobacterium comprising anucleic acid of interest to be incorporated into the genome of thesoybean cells;

[0032] (c) inducing shoot formation from the primary leaf base area;

[0033] (d) cultivating a formed shoot in a medium containing a selectionagent;

[0034] (e) selecting a transformed shoot; and

[0035] (f) regenerating a selected transformed shoot into a soybeanplant.

[0036] In another embodiment, a portion of each of the primary leaves ofthe explant generated in (a)(ii) is removed, thereby generating a pairof primary leaf bases.

[0037] The method of the invention may be employed to introduce anydesired nucleic acid into a soybean cell. In one embodiment of theinvention, the nucleic acid comprises a gene that would express adesirable agronomic trait in soybean.

[0038] In another embodiment of the invention, the nucleic acidcomprises a phosphomannose isomerase gene, which is used as a selectablemarker gene.

[0039] In an additional embodiment of the invention, the co-cultivatingof the explant with Agrobacterium is carried out in the presence ofmannose.

[0040] Both mature and immature seeds may be employed to generate theexplant used in the present invention.

BRIEF DESCRIPTION OF THE FIGURES

[0041]FIG. 1 shows a map of plasmid pNOV2105.

[0042]FIG. 2 shows a map of plasmid pNOV2145.

[0043]FIG. 3 shows a map of plasmid pNOV2147.

[0044]FIG. 4 shows an exemplary process for preparing a soybean explant.Panel A depicts a soybean seed embryo in which a part of the hypocotylis removed. Panel B depicts the soybean explant from Panel A in whichone cotyledon is removed along with its adjacent axillary bud. Panel Cdepicts the soybean explant from Panel B after removal of the twoprimary leaves, generating a break point at the base of each primaryleaf.

[0045]FIG. 5 shows a map of plasmid pBSC11234.

[0046]FIG. 6 shows a map of plasmid pBSC11369.

DETAILED DESCRIPTION

[0047] The present invention will now be described more fullyhereinafter with reference to the accompanying figures, in which variousembodiments of the invention are described. This invention may, however,be embodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete and will fullyconvey the scope of the invention to those skilled in the art. Theterminology used in the description of the invention herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting of the invention. As used in the description of theinvention and the appended claims, the singular forms “a”, “an” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise.

[0048] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs.

[0049] Except as otherwise indicated, standard methods may be used forthe production of cloned genes, expression cassettes, vectors (e.g.,plasmids), proteins and protein fragments, and transformed cells andplants according to the present invention. Except as otherwiseindicated, standard methods may be used for the production of clonedgenes, expression cassettes, vectors (e.g., plasmids), proteins andprotein fragments according to the present invention. Such techniquesare known to those skilled in the art. See e.g., J. Sambrook et al.,Molecular Cloning: A Laboratory Manual Second Edition (Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989), and F. M. Ausubel etal., Current Protocols In Molecular Biology (Green PublishingAssociates, Inc. and Wiley-Interscience, New York, 1991); J. Draper etal., eds., Plant Genetic Transformation And Gene Expression: ALaboratory Manual, (Blackwell Scientific Publications, 1988); and S. B.Gelvin & R. A. Schilperoort, eds., Introduction, Expression, AndAnalysis Of Gene Production In Plants.

[0050] The present invention is drawn to methods and compositions forthe stable transformation of soybean with nucleic acid sequences ofinterest and the regeneration of transgenic soybean plants.

[0051] The methods of the invention may be employed to express anynucleic acid of interest in soybean plants. A gene of interest may be,for example, a gene for herbicide resistance, disease resistance, orinsect/pest resistance, or is a selectable or scorable marker, andcomprises a plant-operable promoter, a coding region, and a 3′terminator region. Herbicide resistance genes include the AHAS gene forresistance to imidazolinone or sulfonyl urea herbicides, the pat or bargene for resistance to bialaphos or glufosinate, the EPSP synthase genefor resistance to glyphosate, etc. Disease resistance genes includegenes for antibiotic synthetic enzymes, e.g., for pyrrolnitrin syntheticenzymes, plant derived resistance genes, and the like. Insect resistancegenes include genes for insecticidal proteins from Bacillusthuringiensis. Genes of interest may also encode enzymes involved inbiochemical pathways, the expression of which alters a trait that isimportant in food, feed, nutraceutical, and/or pharmaceuticalproduction. The gene of interest may be located on a plasmid. A plasmidsuitable for use in the present invention may comprise more than onegene of interest and/or the Agrobacterium may comprise differentplasmids having different genes of interest.

[0052] The present invention provides a method for the transformation ofvarieties of soybean, including Glycine max. The method is based onAgrobacterium-mediated delivery of a desired gene into a soybean cellfollowed by regeneration of transformed cell(s) into a transformedsoybean plant. The methods of the invention are cultivar independent.

[0053] In one embodiment of the invention, an explant is prepared bygerminating a soybean mature seed or immature seed collected from agreenhouse grown plant in a seed germination medium for a period oftime, removing seed coat and, subsequently, a cotyledon from said matureseed or immature seed. In a preferred embodiment of the invention, aportion of the exposed primary leaves is then removed, thereby creatinga break point at the primary leaf base (FIG. 4). Agrobacterium-mediatedgene delivery is made into the cells at the primary leaf base or in thearea of the primary leaf break point. Adventitious shoots are inducedfrom the primary leaf base area of the epicotyl. This induction isachieved by removing pre-existing meristems (i.e., primary, secondary,and axillary meristems) and subjecting the explant to a shoot inductionmedium containing appropriate growth regulators. The shoot inductionprocess facilitates the development or regeneration of transformedshoots from the targeted primary leaf base cells.

[0054] Transformed soybean cells are cultured in the presence of aselection agent. Preferably, the cells are transformed with aphosphomannose isomerase (PMI) gene, and the transformed cells arecultivated in the presence of mannose. In a medium that contains mannoseas a selection agent, soybean cells transformed with a PMI gene have agrowth advantage over those that are not so transformed.

[0055] The time required for regenerating a transformed soybean plantusing the method described in this invention is significantly reducedcompared to other Agrobacterium-mediated transformation protocols thatare reported in the literature. A rooted transformed soybean shoot maybe produced 8 to 12 weeks from the initiation of a transformationexperiment. A foreign genetic construct, or transgene, to be insertedinto the soybean genome is created in vitro by normal techniques ofrecombinant DNA manipulations. The construct may be comprised of anyheterologous nucleic acid. The genetic construct is transformed into theAgrobacterium strain for delivery into the soybean cells. TheAgrobacterium is non-oncogenic, and several such strains are now widelyavailable. The Agrobacterium is preferably selected from A. tumefaciensand A. rhizogenes.

[0056] The foreign genetic construct preferably comprises a selectablemarker gene. The preferred selectable marker gene is a phosphomannoseisomerase gene. Other suitable selectable marker genes include, but arenot limited to, genes encoding: neomycin phosphotransferase II (Fraleyet al., CRC Critical Reviews in Plant Science 4, 1 (1986)); cyanamidehydratase (Maier-Greiner et al., Proc. Natl. Acad. Sci. USA 88, 4250(1991)); aspartate kinase; dihydrodipicolinate synthase (Perl et al.,BioTechnology 11, 715 (1993)); bar gene (Toki et al., Plant Physiol.100, 1503 (1992); Meagher et al., Crop Sci. 36, 1367 (1996));tryptophane decarboxylase (Goddijn et al., Plant Mol. Biol. 22, 907(1993)); neomycin phosphotransferase (NEO; Southern et al., J Mol. Appl.Gen. 1, 327 (1982)); hygromycin phosphotransferase (HPTor HYG; Shimizuet al., Mol. Cell. Biol. 6, 1074 (1986)); dihydrofolate reductase(DHFR); phosphinothricin acetyltransferase (DeBlock et al., EMBO J 6,2513 (1987)); 2,2-dichloropropionic acid dehalogenase(Buchanan-Wollatron et al., J Cell. Biochem. 13D, 330 (1989));acetohydroxyacid synthase (U.S. Pat. No. 4,761,373 to Anderson et al.;Haughn et al., Mol. Gen. Genet. 221, 266 (1988));5-enolpyruvyl-shikimate-phosphate synthase (aroA; Comai et al., Nature317, 741 (1985)); haloarylnitrilase (WO 87/04181 to Stalker et al.);acetyl-coenzyme A carboxylase (Parker et al., Plant Physiol. 92, 1220(1990)); dihydropteroate synthase (sull; Guerineau et al., Plant Mol.Biol. 15, 127 (1990)); and 32 kDa photosystem II polypeptide (psbA;Hirschberg et al., Science 222, 1346 (1983)).

[0057] Also included are genes encoding resistance to chloramphenicol(Herrera-Estrella et al., EMBO J 2, 987 (1983)); methotrexate(Herrera-Estrella et al., Nature 303, 209 (1983); Meijer et al., PlantMol. Biol. 16, 807 (1991)); hygromycin (Waldron et al., Plant Mol. Bio.5, 103 (1985); Zhijian et al., Plant Science 108, 219 (1995); Meijer etal., Plant Mol. Bio. 16, 807 (1991)); streptomycin (Jones et al., Mol.Gen. Genet. 210, 86 (1987)); spectinomycin (Bretagne-Sagnard et al.,Transgenic Res. 5, 131 (1996)); bleomycin (Hille et al., Plant Mol.Biol. 7, 171 (1986)); sulfonamide (Guerineau et al., Plant Mol. Bio. 15,127 (1990); bromoxynil (Stalker et al., Science 242, 419 (1988)); 2,4-D(Streber et al., Bio/Technology 7, 811 (1989)); phosphinothricin(DeBlock et al., EMBO J. 6, 2513 (1987)); spectinomycin(Bretagne-Sagnard and Chupeau, Transgenic Research 5, 131 (1996)).

[0058] In one embodiment, the starting material for the transformationprocess is a soybean mature seed. In another embodiment, the startingmaterial can be a soybean immature seed from a growing soybean plant.The seed is placed on a germination medium and permitted to germinatefor a period of 6-24 hours, preferably for about 6-14 hours, and morepreferably for about 8-12 hours. Seeds may also be allowed to germinatefor a longer period of time, for example, from 2 to 5 days, if desired.

[0059] The seed coat and hypocotyl of the germinating seed is removed.One cotyledon along with its adjacent axillary shoot bud is alsoremoved. Afterwards, the primary leaves are substantially removed,thereby creating an explant comprising the primary leaf base, epicotylto which the leaf base is attached, and a cotyledon to which theepicotyl is attached. Substantially removed means removal of a majorportion of primary leaf tissue.

[0060] For Agrobacterium-mediated gene transfer, wounding of the planttissue is known to facilitate gene transfer. Therefore it is preferred,but not necessary, that a wound is created at the leaf base region.

[0061] The explant, prepared as described above, is then immersed intoan Agrobacterium cell suspension for a few minutes to a few hours,typically about 0.5-3 hours, and preferably 1-2 hours. ExcessiveAgrobacterium cell suspension is removed and the remaining Agrobacteriumare permitted to co-cultivate with the explant on a co-cultivationmedium for several days, typically two to five days, and preferablythree to four days, under 16h light/8h dark conditions at a temperatureof about 22° C.±2° C.

[0062] After co-cultivation, the explant is transferred to a medium (ora series of media) conducive to shoot development and selection oftransformed cells, for 8-12 weeks. Such a medium (or media) generallycontains a shoot-inducing hormone as well as a selection agent. Theregeneration media used in the examples below contain mannose, as theselection agent, as well as benzylaminopurine (BAP), a shoot-inducinghormone. The term hormone also includes cell growth regulating compoundsthat induce shoot formation, including, but-not limited to, auxins (suchas, e.g., IAA, NAA, and indole butyric acid (IBA)), cytokinins (such as,e.g., thidiazuron, kinetin, and isopentenyl adenine), and/or gibberellicacids (GA₃).

[0063] When shoots reach about 2 cm and with full trifoliate leafformation, shoots are separated from the explant and placed on a rootingmedium to induce root formation. Preferably, the rooting medium alsocontains a selection agent to further help identify potentialtransformed shoots. Root formation takes approximately 1-2 weeks,following which the plants can be transferred to soil and grown to fullmaturity.

[0064] Transgenic plants comprising a heterologous nucleic acid (i.e.,comprising cells or tissues transformed in accordance with the methodsdescribed herein), as well as the seeds and progeny produced by thetransgenic plants, are an additional aspect of the present invention.Procedures for cultivating transformed cells to useful cultivars areknown to those skilled in the art. Techniques are known for the in vitroculture of plant tissue, and in a number of cases, for regeneration intowhole plants. A further aspect of the invention is transgenic planttissue, plants, or seeds containing the nucleic acids described above.In a preferred embodiment, transformed plants produced using the methodsdescribed herein are not chimeric, or only a small proportion oftransformed plants is chimeric. This is preferably achieved by extendingthe period of high cytokinin treatment or by increasing the stringencyof mannose selection, or both.

[0065] Thus, the transformed cells of the present invention, identifiedby selection or screening and cultured in an appropriate medium thatsupports regeneration as provided herein, may then be allowed to matureinto plants. Plants are preferably matured either in a growth chamber orgreenhouse. Plants are regenerated from about 2-6 weeks after atransformant is identified, depending on the initial tissue. Duringregeneration, cells may be grown on solid media in tissue culturevessels. Illustrative embodiments of such vessels are petri dishes andPlant Con®s. After the regenerating plants have reached the stage ofshoot and root development, they may be transferred to a greenhouse forfurther growth and testing. As provided above, seeds and progeny plantsof the regenerated plants are an aspect of the present invention.Accordingly, the term “seeds” is meant to encompass seeds of thetransformed plant, as well as seeds produced from the progeny of thetransformed plants. Plants of the present invention include not only thetransformed and regenerated plants, but also progeny of transformed andregenerated plants produced by the methods described herein.

[0066] Plants produced by the described methods may be screened forsuccessful transformation by standard methods described above. Seeds andprogeny plants of regenerated plants of the present invention may becontinuously screened and selected for the continued presence of thetransgenic and integrated nucleic acid sequence in order to developimproved plant and seed lines, which are another aspect of the presentinvention. Desirable transgenic nucleic acid sequences may thus be moved(i.e., introgressed or inbred) into other genetic lines such as certainelite or commercially valuable lines or varieties. Methods ofintrogressing desirable nucleic acid sequences into genetic plant linesmay be carried out by a variety of techniques known in the art,including by classical breeding, protoplast fusion, nuclear transfer andchromosome transfer. Breeding approaches and techniques are known in theart, and are set forth in, for example, J. R. Welsh, Fundamentals ofPlant Genetics and Breeding (John Wiley and Sons, New York, (1981));Crop Breeding (D. R. Wood, ed., American Society of Agronomy, Madison,Wis., (1983)); O. Mayo, The Theory of Plant Breeding, Second Edition(Clarendon Press, Oxford, England (1987)); and Wricke and Weber,Quantitative Genetics and Selection Plant Breeding (Walter de Gruyterand Co., Berlin (1986)). Using these and other techniques in the art,transgenic plants and inbred lines obtained according to the presentinvention may be used to produce commercially valuable hybrid plants andcrops, which hybrids are also an aspect of the present invention.

[0067] The foregoing is illustrative of the various embodiments of thepresent invention and is not to be construed as limiting thereof.

[0068] The invention will be further described by the followingexamples, which are not intended to limit the scope of the invention inany manner.

EXAMPLE 1

[0069] Transformation Vectors

[0070] The plasmid pNOV2105 (FIG. 1) is a modification of pVictor, whichis disclosed and described in WO 97/04112 in that the 35S promoter isreplaced with a SMAS promoter, the 35S terminator is replaced with theNos terminator, and an additional SMAS promoter is inserted upstream ofthe GUSintronGUS sequence, which is flanked on its 3′ end by a Nosterminator. pNOV2105 employed in the methods described herein does notcontain the multicloning site that is found in pVictor. However, it iswell within the skill in the art to add such a cloning site, if desired.

[0071] pNOV2105 (FIG. 1) is a vector for Agrobacterium-mediated planttransformation and contains the Ti right and left border sequences fromthe nopaline type pTiT37 plasmid (Yadav et al. 1982 Proc Natl Acad Sci79:6322-6326) flanking the genes phosphomannose isomerase (PMI) andbeta-glucoronidase (GUS).

[0072] For replication and maintenance in E. coli, the plasmid containsthe origin of replication from the E. coli plasmid pUC19 (pUC19ori)(Yanish-Perron et al. 1985 Gene 33:103-119), and for replication andmaintenance in Agrobacterium tumefaciens the plasmid further containsthe origin of replication from the Pseudomonas plasmid pVS1 (pVSlori)(Itoh et al. 1984 Plasmid 11:206-220; Itoh and Haas 1985 Gene 36:27-36).For selection in E. coli and Agrobacterium tumefaciens, the plasmidcontains the spectinomycin/streptomycin resistance gene (spec/strep)from the transposon Tn7 encoding the enzyme3″(9)-O-nucleotidyltransferase (Fling et al. 1985 Nucleic Acids Res19:7095-7106). The spec/strep resistance gene is fused to the tacpromoter (see, e.g., Amann et al. 1983 Gene 25(203):167-78) forefficient expression in the bacterium.

[0073] The T-DNA segment between the right and left border harbors thefollowing genes, which are the only genes transferred to the soybeanplant via the Agrobacterium tumefaciens-mediated transformation.

[0074] GUSintronGUS

[0075] beta-glucuronidase (GUS): This segment next to the right bordercontains the beta-glucuronidase gene (GUS) from E. coli with an intronin the coding region to prevent translation by Agrobacterium fused tothe SMAS promoter and Nos terminator. The GUSintronGUS gene was isolatedfrom plasmid pBISN1. (Narasimhulu et al. 1986 Early transcription ofAgrobacterium DNA in tobacco and maize, Plant Cell 8:873-866).

[0076] phosphomannose isomerase (PMI): This segment next to the leftborder is the mannose-6-phosphate isomerases gene from E. coli (Milesand Guest 1984, Gene 32:41-48) fused to the SMAS promoter (Ni M, Cui D,Einstein J, Narasimhulu S, Vergara CE, Gelvin SB (1995) and Nosterminator. The phosphomannose isomerase gene is used as a selectionmarker to select transgenic shoots on media containing D-mannose as thecarbon source.

[0077] The components and sequence of pNOV2145 (FIG. 2) are set forth inSEQ ID NO: 1. The components and sequence of pNOV2147 (FIG. 3) are setforth in SEQ ID NO: 2.

EXAMPLE 2

[0078] Transformation and Regeneration

[0079] Mature dried soybean seeds (Var. S42 HI) were surface sterilizedby releasing chlorine gas inside a desiccator. Seeds were kept in petriplates and chlorine gas was produced by pouring 100 ml of Cloroxg into abeaker and slowly adding 8 ml of concentrated HCl. Seeds were sterilizedby at least two gas release treatments each lasting for 8-18 hours.

[0080] Sterilized seeds (approximately 15-20 seeds per plate) were thenplaced on a germination medium containing 0.6% agar-solidified MS basalmedium (Murashige and Skoog (1962) A revised medium for rapid growth andbioassays with tobacco callus cultures. Physiol Plant 15: 473-479) and2% sucrose. The pH was maintained at 5.8. The petri plates were placedin a room at 37° C. for overnight growth or imbibition of seeds. Theseed coat was removed, followed by removing part of the hypocotyl,keeping about 0.5 cm of the hypocotyl. One cotyledon was removed alongwith its adjacent axillary shoot bud and was discarded. On the remainingcotyledon, the primary leaves were broken apart using a scalpel, leavingthe primary leaf bases on the epicotyl. (FIG. 4)

[0081] Agrobacterium strain (LBA 4404) containing the plasmid pNOV 2145(ZsGreen1 and PMI, as described in Example 1) was streaked from frozenglycerol stocks onto YEP plates (yeast extract 10 g/L, peptone 5 g/L,NaCl 5 g/L, bacto agar 15 g/L) containing appropriate antibiotic (100mg/L spectinomycin). Agrobacterium was then incubated at 27° C. for 1-2days. A scoop of Agrobacterium from plates were grown on 100 ml YEPliquid medium containing an antibiotic (100 mg/L spectinomycin) forovernight growth at 27° C. on a shaker. Bacterial suspensions werecentrifuged at about 1500 g for 15 minutes and resuspended to a densityof OD ₆₆₀=0.2 or 0.65 in a co-cultivation liquid medium (B₅ salts 0.05X(Sigma), B₅ vitamins (0.05X) (B₅ vitamin composition (1X): inositol 100mg/L, nicotinic acid 1 mg/L, pyridoxine HCl 1 mg/L, thiamine HCl 10mg/L), acetosyringone 40 mg/L, sucrose 20 g/L, BAP 2 mg/L, GA₃ 0.25mg/L, MES (Morpholino ethanesulfonic acid) 3.9 g/L, and pH 5.4.

[0082] The explants containing the target tissue were immersed intoAgrobacterium suspension and incubated for 1-2 hours. The Agrobacteriumsuspension was poured off, and the treated explants were placed onto afilter paper inside co-cultivation plates. The adaxial side of theexplants was kept in contact with the filter paper. The co-cultivationsolid medium was composed of B₅ salts (Sigma, 0.05X), B₅ vitamins(0.05X), 40 mg/L acetosyringone, sucrose 20 g/L, BAP 2 mg/L, GA₃ 0.25mg/L, MES 3.9 g/L, and pH 5.4. The medium was solidified with 0.5%purified agar (Sigma).

[0083] The explants were co-cultivated with the Agrobacterium at 20-23°C. for a period of 2-5 days, under 16 h light/8 h dark conditions. Afterco-cultivation, the explants were washed in sterile water containing 250mg/L cefotaxime, primary and secondary meristems were removed, and theexplants were transferred to regeneration medium (i.e., REG-1 medium).During the regeneration process, any axillary shoots adjacent to thecotyledon were also removed to encourage growth from the area of theprimary leaf base.

[0084] REG-1 medium contained MS salts (1X), B₅ vitamins (1X), KNO₃ ₁g/L, BAP 1 mg/L, ticarcillin 300 mg/L, cefotaxime 100 mg/L, glutamine250 mg/L, asparagine 50 mg/L, mannose 15-30 g/L, sucrose 0, 0.25, and 1g/L, pH 5.6, and purified agar 10 g/L. Five explants were placed in eachpetri plate in an upright position, such that the epicotyl end of theexplant was inserted into the medium. The plates were kept inside aplastic container and placed in a culture room at 22-25° C., under an18-20 hr light/4-6 hr dark cycle at 60-100 μE m⁻² S⁻¹. After 2 weeks onREG-1 medium, explants were transferred to REG-2 medium, which containedMS salts (1X) and B₅ vitamins (1X), KNO₃ 1 g/L, BAP 0.5 mg/L,ticarcillin 300 mg/L, cefotaxime 100 mg/L, glutamine 250 mg/L,asparagine 50 mg/L, mannose 15 g/L, and sucrose 1 g/L. The media pH wasmaintained at 5.6, and the media was solidified with purified agar 10g/L.

[0085] At 4-6 weeks, the soybean cultures were transferred to REG-3medium for continuing selection and shoot development. REG-3 mediumcontained MS salts (1X), B₅ vitamins (1X), KNO₃ 1 g/L, BAP 0.2 mg/L, GA₃0.5 mg/L, IBA 0.1 mg/L, ticarcillin 300 mg/L, cefotaxime 100 mg/L,glutamine 250 mg/L, asparagine 50 mg/L, mannose 15 g/L, sucrose 1 g/L,pH 5.6, and the medium was solidified with purified agar 10 g/L. Deadtissue was removed and explants with regenerating shoots weresubcultured in fresh REG-3 medium every two weeks. Elongated shoots werecontinuously harvested from the cultures when they reached about 2-4 cmin length. At that time, shoots were transferred to a rooting medium,which contained MS salts (0.5X), B₅ Vitamins (0.5X), glutamine 250 mg/L,asparagine 50 mg/L, KNO3 1 g/L, cefotaxime 100 mg/L, ticarcillin 300mg/L, sucrose 15 g/L, IBA 0.5 mg/L, pH 5.6, and purified agar 10 g/L.

[0086] Rooted transgenic shoots expressing a fluorescent protein gene(ZsGreen1) were transferred to 2″ pots which contained moistened Fafardgerminating mix (Conrad Fafard Inc., MA, USA) and were kept covered withplastic cups for maintaining moisture for approximately 2 weeks. Plantswere acclimatized at 27-29° C. day temperature, 21° C. nighttemperature, and a 16 h photoperiod (20-40 μE m⁻²S⁻¹ light intensity).When new leaves began to emerge, plants were transferred to one-gallonpots which contained a soil mixture composed of 50-55% composted pinebark, 40-45% Peat, 5-10% Perlite (Sungrow Horticultural Supply, PineBluff, Ark.). Acclimatized soybean plants were grown in the greenhouseat 27-29° C. day temp, 21° C. night temp, 400-600 μE m⁻² S⁻¹ lightintensity, 70-95% relative humidity, and a 16 hr photoperiod. The plantswere fertilized with osmocote (Scotts-Sierra Horticultural ProductsCompany, Ohio; 17-6-12) twice (5-8 g/gallon soil) during the growthperiod. Transformation was confirmed by Taqman analysis for the presenceof the fluorescent protein gene as well as the PMI gene in the leaves ofthe greenhouse grown plants. Expression of the fluorescent protein genein the transformed soybean tissue was also confirmed by visualizing theexpression using a fluorescent microscope.

[0087] Six transgenic plants developed using the gene construct pNOV2145were confirmed by Southern blot analyses. Progeny analysis of one eventfor either the PMI gene or the ZsGreen1 gene revealed one integrationsite of the T-DNA into the genome of the transformed soybean, and theprogeny segregated in a 3:1 ratio in the T1 generation. TABLE 1Transformed shoots expressing fluorescent protein gene (Zsgreen1)Transformed Expt No. Gene construct shoots/explant Percent tr. shoots 75pNOV2145 5/45 11 89 pNOV2145 5/75  7

EXAMPLE 3

[0088] Soybean seeds (Var. S42 Hi) were surface sterilized and explantswere prepared as described in Example 2.

[0089] Agrobacterium strain (LBA 4404) carrying the plasmid pNOV2147 wasprepared as described in Example 1. The final bacterial concentrationwas adjusted to OD ₆₆₀=0.60 with a co-cultivation liquid medium. Theconditions for explant preparation, Agrobacterium inoculation, andco-cultivation were the same as those described in Example 2.

[0090] Following three days of co-cultivation in a solid co-cultivationmedium, excessive Agrobacterium was washed off, primary and secondarymeristems were removed, and the explants were transferred to REG-1medium. They were cultured at 28-30° C. in 16 h light and 8 h darkconditions. After 2 weeks on REG-1 medium, the cultures were transferredto REG-2 medium. During this regeneration process, only shoots arisingfrom the base of a primary leaf were kept. At about the 4th week, theshoot cultures were transferred to REG-3 medium. They were thentransferred to fresh REG-3 medium every 10-14 days. As in REG-1 andREG-2 medium, only the new shoots arising from the base of a primaryleaf were kept while the rest of the shoots were removed. When elongatedshoots reached about 2-4 cm in length, they were separated from the restof the shoot cultures and transferred to rooting medium.

[0091] Five transgenic shoots out of 35 explants were identified asexpressing the cyano fluorescent protein gene (Table 2). TABLE 2Transformed shoots expressing the cyano fluorescent protein geneTransformed % Experiment No. Gene construct shoots/explants transformedshoots 92 pNOV2147 5/35 14

EXAMPLE 4

[0092] Mannose Treatment During Co-cultivation

[0093] The gene construct used in this example was pNOV2145 (whichcomprises ZsGreen1 and PMI genes, as described in Example 1). Theprocedures for preparing the explants, Agrobacteria suspensions, andinoculation of explants with bacterial suspensions were carried out asdescribed in Example 2. The final bacterial concentration was adjustedto OD ₆₆₀ =0.55 or 0.85.

[0094] Following the inoculation step, explants were transferred to aco-cultivation medium containing either 20 g/L sucrose or 15 g/L mannoseand were kept at 20-23° C. under 16 h light and 8 h dark conditions.

[0095] After 3-5 days of co-cultivation, expression of the fluorescentprotein gene was visualized using a fluorescent microscope. Explantsthat were inoculated with Agrobacterium in a mannose-containingco-cultivation medium showed at least two-fold the number of fluorescentspots compared to those co-cultivated in a sucrose-containingco-cultivation medium. Subsequent shoot regeneration and selection stepswere followed as those described in Example 2.

[0096] A significant increase in the production of transformed shootswas observed in the experiments where mannose was included in theco-cultivation medium (Table 3). Five transformed shoots fromco-cultivation medium that included mannose were rooted and transferredto soil. Subsequent analysis by Taqman as well as Southern blotconfirmed the integration of the transgenes. Transgene expression in theT1 progeny confirmed the germline transmission of the transgenes. TABLE3 Transformed shoots expressing ZsGreen1 fluorescent protein gene whereexplants and Agrobacteria were co-cultivated in mannose or sucroseExperi- Gene Co-culture in Transformed % Trans- ment No. constructmannose/sucrose shoots/explants formation  87 pNOV2145 Sucrose 0/60 0Mannose 6/80 7.5 102 pNOV2145 Sucrose 1/20 5 Mannose 8/40 20

EXAMPLE 5

[0097] In this example, Agrobacterium EHA101 comprising the plasmidpNOV2105 (SMAS-PMI SMAS-GUS, as described in Example 1) was used insoybean transformation. The preparation of the explants, Agrobacteriasuspension, and inoculation of explants with Agrobacteria were the sameas those described in Example 2. The final bacterial concentration wasadjusted to OD ₆₆₀=0.45 or 0.6.

[0098] Following Agrobacterium inoculation, explants were transferred toa co-cultivation medium containing either 20 g/L sucrose or 15 g/Lmannose. Co-cultivation was carried out at 20-23° C. under a 16 h lightand 8 h dark conditions. Following 3-5 days of co-cultivation, GUS geneexpression was visualized using a histochemical gus assay. Explantsco-cultivated in mannose-containing co-cultivation medium showed atleast two-fold the number of GUS spots compared to those co-cultivatedin sucrose-containing co-cultivation medium. Shoot regeneration andselection were carried out as described in Example 2. A significantincrease in the production of transformed shoots was observed in theexperiment in which mannose was added into the co-cultivation medium.(Table 4). TABLE 4 Transformed shoots expressing GUS gene Experi- GeneCo-cultivation in Transformed % Trans- ment No. constructmannose/sucrose shoots/explant formation 63 pNOV2105 sucrose 5/60  8 81pNOV2105 Sucrose 2/30  7 Mannose 5/30 17

EXAMPLE 6

[0099] In this example, Agrobacterium EHA101 comprising the plasmidpBSC11234 (FIG. 5) was used in soybean transformation. The componentsand sequence of pBSC11234 are set forth in SEQ ID NO:3. pBSC11234comprises a CMP-PMI : beta conglycinin-galactosidase gene construct. Thepreparation of the explants, Agrobacteria suspension, and inoculation ofexplants with Agrobacteria were the same as those described in Example2. The final bacterial concentration was adjusted to OD ₆₆₀=0.6. Theco-cultivation liquid medium contained B5 salts (0.1X), B5 vitamins(1X), acetosyringone 80 mg/L, sucrose 20 g/L, BAP 2 mg/L, GA₃ 0.25 mg/L,MES 3.9 g/L, and pH 5.4. Solid co-cultivation medium was prepared byincorporating 5 g/L purified agar to the liquid co-cultivation medium.

[0100] Following Agrobacterium inoculation, explants were transferred toa solid co-cultivation medium and cultured at 20-24° C. under 16 h lightand 8 h dark conditions. Following 3-5 days of co-cultivation, primaryand secondary shoot meristems were removed and discarded, and theresulting explants were transferred to REG-4 medium, which contained B5salts (1X), B5 Vitamins (1X), BAP 1 mg/L, glutamine 50 mg/L, asparagine50 mg/L, cefotaxime 100 mg/L, ticarcillin 300 mg/L, mannose 15-20 g/L,sucrose 0, 0.25, or 1 g/L, purified agar 10 g/L, and pH at 5.6. After aperiod of 5-7 days, any shoot grown from the axillary meristem close tothe cotyledon was removed, and the explants were transferred to REG-5medium, which contained B₅ salts (1X), B₅ Vitamins (1X), BAP 0.5 mg/L,glutamine 50 mg/L, asparagine 50 mg/L, cefotaxime 100 mg/L, ticarcillin300 mg/L, mannose 15 g/L, sucrose 1 g/L, purified agar 10 g/L, and pH at5.6.

[0101] At four weeks, explants were transferred to REG-6 medium forelongation of shoots. REG-6 medium contained MS salts (1X), MS Vitamins(1X) (MS vitamin composition: inositol 100 mg/L, nicotinic acid 0.5mg/L, pyridoxine HCl 0.5 mg/L, thiamine HCl 0.1 mg/L, glycine 2 mg/L),myo-inositol 200 mg/L, BAP 0.2 mg/L, zeatin riboside 0.5 mg/L, IBA 0.1mg/L, GA₃ 1 mg/L, glutamine 50 mg/L, asparagine 50 mg/L, ticarcillin 300mg/L, mannose 15 g/L, sucrose 5 g/L, silver nitrate 0.8 mg/L, purifiedagar 10 g/L, and pH 5.6. Explants were transferred to fresh REG-6 mediumevery two weeks. Elongated shoots (2-4 cm long) were removed and rootedin rooting medium and transferred to soil. The rooting medium containedMS salts (1X), B₅ Vitamins (1X), glutamine 100 mg/L, asparagine 100mg/L, IBA 0.7 mg/L, timentin 100 mg/L, and sucrose 15 g/L. Taqmananalysis confirmed the presence of the transgenes (alpha galactosidaseand phosphomannose isomerase) in leaf samples from two events.

EXAMPLE 7

[0102] In this example, Agrobacterium EHA101 comprising the plasmidpBSC11369 (FIG. 6) was used in soybean transformation. The componentsand sequence of pBSC11369 are set forth in SEQ ID NO: 4. pBSC11369comprises a CMP-HPT: CMP-ZsGreen1 gene construct. The preparation of theexplants, Agrobacteria suspension, and inoculation of explants withAgrobacteria were the same as those described in Example 2. The finalbacterial concentration was adjusted to OD ₆₆₀ =0.6. The co-cultivationliquid medium contained B₅ salts (0.1X), B₅ vitamins (1X),acetosyringone 80 mg/L, sucrose 20 g/L, BAP 2 mg/L, GA₃ 0.25 mg/L, MES3.9 g/L, and pH 5.4. Solid co-cultivation medium was prepared byincorporating 5 g/L purified agar to the liquid co-cultivation medium.

[0103] Following Agrobacterium inoculation, explants were transferred toa solid co-cultivation medium and cultured at 20-24° C. under 16 h lightand 8 h dark conditions. Following 3-5 days of co-cultivation, explantswere transferred to REG-7 medium after removing primary and secondarymeristems from the explants in order to encourage shoot growth from theprimary leaf base area. REG-7 medium contained B₅ salts (1X), B₅Vitamins (1X), BAP 1 mg/L, glutamine 50 mg/L, asparagine 50 mg/L,cefotaxime 100 mg/L, ticarcillin 300 mg/L, sucrose 30 g/L, hygromycin2-5 mg/L, purified agar 10 g/L, and pH 5.6. Explants were placed in anupright position such that the epicotyl end of the explant was insertedinto the medium. After a period of 7-10 days, any shoots grown from theaxillary meristem close to the cotyledon were removed. Explants weretransferred to fresh REG-8 medium, which contained B₅ salts (1X), B₅Vitamins (1X), BAP 0.5 mg/L, glutamine 50 mg/L, asparagine 50 mg/L,cefotaxime 100 mg/L, ticarcillin 300 mg/L, sucrose 30 g/L, purified agar10 g/L, and pH at 5.6. After another two weeks, explants weretransferred to RLG-9 medium and subcultured thereafter every two weeks.REG-9 medium contained MS salts (1X), MS Vitamins (1X), myo-inositol 200mg/L, BAP 0.2 mg/L, zeatin riboside 0.5 mg/L, IBA 0.1 mg/L, GA₃ 1 mg/L,glutamine 50 mg/L, asparagine 50 mg/L, silver nitrate 0.8 mg/L,ticarcillin 300 mg/L, sucrose 30 g/L, hygromycin 0.1-0.2 mg/L, purifiedagar 10 g/L, and pH 5.6. Elongated shoots (2-4 cm long) were removed,rooted in rooting medium, and then transferred to soil. The rootingmedium contained MS salts (1X), B₅ Vitamins (1X), glutamine 100 mg/L,asparagine 100 mg/L, IBA 0.7 mg/L, timentin 100 mg/L, and sucrose 15g/L. Taqman analysis confirmed the presence of the, transgenes (HPT aswell as ZsGreen1) in leaf samples obtained from five events. Expressionof the ZsGreen1 gene in plant parts was confirmed by visualization undera fluorescent microscope.

[0104] All publications, patents, and patent applications cited hereinare incorporated by reference. While in the foregoing specification thisinvention has been described in relation to certain preferredembodiments thereof, and many details have been set forth for purposesof illustration, it will be apparent to those skilled in the art thatthe invention is susceptible to additional embodiments and that certainof the details described herein may be varied considerably withoutdeparting from the basic principles of the invention.

1 4 1 9555 DNA Artificial pNOV2145 1 gatccaccgg tcgccaccat ggcccagtccaagcacggcc tgaccaagga gatgaccatg 60 aagtaccgca tggagggctg cgtggacggccacaagttcg tgatcaccgg cgagggcatc 120 ggctacccct tcaagggcaa gcaggccatcaacctgtgcg tggtggaggg cggccccttg 180 cccttcgccg aggacatctt gtccgccgccttcatgtacg gcaaccgcgt gttcaccgag 240 tacccccagg acatcgtcga ctacttcaagaactcctgcc ccgccggcta cacctgggac 300 cgctccttcc tgttcgagga cggcgccgtgtgcatctgca acgccgacat caccgtgagc 360 gtggaggaga actgcatgta ccacgagtccaagttctacg gcgtgaactt ccccgccgac 420 ggccccgtga tgaagaagat gaccgacaactgggagccct cctgcgagaa gatcatcccc 480 gtgcccaagc agggcatctt gaagggcgacgtgagcatgt acctgctgct gaaggacggt 540 ggccgcttgc gctgccagtt cgacaccgtgtacaaggcca agtccgtgcc ccgcaagatg 600 cccgactggc acttcatcca gcacaagctgacccgcgagg accgcagcga cgccaagaac 660 cagaagtggc acctgaccga gcacgccatcgcctccggct ccgccttgcc ctgagcggcc 720 ctctagatcc ccgaatttcc ccgatcgttcaaacatttgg caataaagtt tcttaagatt 780 gaatcctgtt gccggtcttg cgatgattatcatataattt ctgttgaatt acgttaagca 840 tgtaataatt aacatgtaat gcatgacgttatttatgaga tgggttttta tgattagagt 900 cccgcaatta tacatttaat acgcgatagaaaacaaaata tagcgcgcaa actaggataa 960 attatcgcgc gcggtgtcat ctatgttactagatcgggaa ttgggtaccg aattcactgg 1020 ccgtcgtttt acaacgtcgt gactgggaaaaccctggcgt tacccaactt aatcgccttg 1080 cagcacatcc ccctttcgcc agctggcgtaatagcgaaga ggcccgcacc gatcgccctt 1140 cccaacagtt gcgcagcctg aatggcgaatggcgcctgat gcggtatttt ctccttacgc 1200 atctgtgcgg tatttcacac cgcatatggtgcactctcag tacaatctgc tctgatgccg 1260 catagttaag ccagccccga cacccgccaacacccgctga cgcgccctga cgggcttgtc 1320 tgctcccggc atccgcttac agacaagctgtgaccgtctc cgggagctgc atgtgtcaga 1380 ggttttcacc gtcatcaccg aaacgcgcgagacgaaaggg cctcgtgata cgcctatttt 1440 tataggttaa tgtcatgata ataatggtttcttagacgtc aggtggcact tttcggggaa 1500 atgtgcgcgg aacccctatt tgtttatttttctaaataca ttcaaatatg tatccgctca 1560 tgagacaata accctgataa atgcttcaatggcgcgccgg taccagcttg catgcctgca 1620 ggtcgactct agaggatcct ggcagacaaagtggcagaca tactgtccca caaatgaaga 1680 tggaatctgt aaaagaaaac gcgtgaaataatgcgtctga caaaggttag gtcggctgcc 1740 tttaatcaat accaaagtgg tccctaccacgatggaaaaa ctgtgcagtc ggtttggctt 1800 tttctgacga acaaataaga ttcgtggccgacaggtgggg gtccaccatg tgaaggcatc 1860 ttcagactcc aataatggag caatgacgtaagggcttacg aaataagtaa gggtagtttg 1920 ggaaatgtcc actcacccgt cagtctataaatacttagcc cctccctcat tgttaaggga 1980 gcaaaatctc agagagatag tcctagagagagaaagagag caagtagcct agaagtagga 2040 tccccgatca tgcaaaaact cattaactcagtgcaaaact atgcctgggg cagcaaaacg 2100 gcgttgactg aactttatgg tatggaaaatccgtccagcc agccgatggc cgagctgtgg 2160 atgggcgcac atccgaaaag cagttcacgagtgcagaatg ccgccggaga tatcgtttca 2220 ctgcgtgatg tgattgagag tgataaatcgactctgctcg gagaggccgt tgccaaacgc 2280 tttggcgaac tgcctttcct gttcaaagtattatgcgcag cacagccact ctccattcag 2340 gttcatccaa acaaacacaa ttctgaaatcggttttgcca aagaaaatgc cgcaggtatc 2400 ccgatggatg ccgccgagcg taactataaagatcctaacc acaagccgga gctggttttt 2460 gcgctgacgc ctttccttgc gatgaacgcgtttcgtgaat tttccgagat tgtctcccta 2520 ctccagccgg tcgcaggtgc acatccggcgattgctcact ttttacaaca gcctgatgcc 2580 gaacgtttaa gcgaactgtt cgccagcctgttgaatatgc agggtgaaga aaaatcccgc 2640 gcgctggcga ttttaaaatc ggccctcgatagccagcagg gtgaaccgtg gcaaacgatt 2700 cgtttaattt ctgaatttta cccggaagacagcggtctgt tctccccgct attgctgaat 2760 gtggtgaaat tgaaccctgg cgaagcgatgttcctgttcg ctgaaacacc gcacgcttac 2820 ctgcaaggcg tggcgctgga agtgatggcaaactccgata acgtgctgcg tgcgggtctg 2880 acgcctaaat acattgatat tccggaactggttgccaatg tgaaattcga agccaaaccg 2940 gctaaccagt tgttgaccca gccggtgaaacaaggtgcag aactggactt cccgattcca 3000 gtggatgatt ttgccttctc gctgcatgaccttagtgata aagaaaccac cattagccag 3060 cagagtgccg ccattttgtt ctgcgtcgaaggcgatgcaa cgttgtggaa aggttctcag 3120 cagttacagc ttaaaccggg tgaatcagcgtttattgccg ccaacgaatc accggtgact 3180 gtcaaaggcc acggccgttt agcgcgtgtttacaacaagc tgtaagagct tactgaaaaa 3240 attaacatct cttgctaagc tgggagctctagatccccga atttccccga tcgttcaaac 3300 atttggcaat aaagtttctt aagattgaatcctgttgccg gtcttgcgat gattatcata 3360 taatttctgt tgaattacgt taagcatgtaataattaaca tgtaatgcat gacgttattt 3420 atgagatggg tttttatgat tagagtcccgcaattataca tttaatacgc gatagaaaac 3480 aaaatatagc gcgcaaacta ggataaattatcgcgcgcgg tgtcatctat gttactagat 3540 cgggaattgg gtaccatgcc cgggcggccagcatggccgt atccgcaatg tgttattaag 3600 ttgtctaagc gtcaatttgt ttacaccacaatatatcctg ccaccagcca gccaacagct 3660 ccccgaccgg cagctcggca caaaatcaccactcgataca ggcagcccat cagaattaat 3720 tctcatgttt gacagcttat catcgactgcacggtgcacc aatgcttctg gcgtcaggca 3780 gccatcggaa gctgtggtat ggctgtgcaggtcgtaaatc actgcataat tcgtgtcgct 3840 caaggcgcac tcccgttctg gataatgttttttgcgccga catcataacg gttctggcaa 3900 atattctgaa atgagctgtt gacaattaatcatccggctc gtataatgtg tggaattgtg 3960 agcggataac aatttcacac aggaaacagaccatgaggga agcgttgatc gccgaagtat 4020 cgactcaact atcagaggta gttggcgtcatcgagcgcca tctcgaaccg acgttgctgg 4080 ccgtacattt gtacggctcc gcagtggatggcggcctgaa gccacacagt gatattgatt 4140 tgctggttac ggtgaccgta aggcttgatgaaacaacgcg gcgagctttg atcaacgacc 4200 ttttggaaac ttcggcttcc cctggagagagcgagattct ccgcgctgta gaagtcacca 4260 ttgttgtgca cgacgacatc attccgtggcgttatccagc taagcgcgaa ctgcaatttg 4320 gagaatggca gcgcaatgac attcttgcaggtatcttcga gccagccacg atcgacattg 4380 atctggctat cttgctgaca aaagcaagagaacatagcgt tgccttggta ggtccagcgg 4440 cggaggaact ctttgatccg gttcctgaacaggatctatt tgaggcgcta aatgaaacct 4500 taacgctatg gaactcgccg cccgactgggctggcgatga gcgaaatgta gtgcttacgt 4560 tgtcccgcat ttggtacagc gcagtaaccggcaaaatcgc gccgaaggat gtcgctgccg 4620 actgggcaat ggagcgcctg ccggcccagtatcagcccgt catacttgaa gctaggcagg 4680 cttatcttgg acaagaagat cgcttggcctcgcgcgcaga tcagttggaa gaatttgttc 4740 actacgtgaa aggcgagatc accaaagtagtcggcaaata aagctctagt ggatctccgt 4800 acccccgggg gatctggctc gcggcggacgcacgacgccg gggcgagacc ataggcgatc 4860 tcctaaatca atagtagctg taacctcgaagcgtttcact tgtaacaacg attgagaatt 4920 tttgtcataa aattgaaata cttggttcgcatttttgtca tccgcggtca gccgcaattc 4980 tgacgaactg cccatttagc tggagatgattgtacatcct tcacgtgaaa atttctcaag 5040 cgctgtgaac aagggttcag attttagattgaaaggtgag ccgttgaaac acgttcttct 5100 tgtcgatgac gacgtcgcta tgcggcatcttattattgaa taccttacga tccacgcctt 5160 caaagtgacc gcggtagccg acagcacccagttcacaaga gtactctctt ccgcgacggt 5220 cgatgtcgtg gttgttgatc taaatttaggtcgtgaagat gggctcgaga tcgttcgtaa 5280 tctggcggca aagtctgata ttccaatcataattatcagt ggcgaccgcc ttgaggagac 5340 ggataaagtt gttgcactcg agctaggagcaagtgatttt atcgctaagc cgttcagtat 5400 cagagagttt ctagcacgca ttcgggttgccttgcgcgtg cgccccaacg ttgtccgctc 5460 caaagaccga cggtcttttt gttttactgactggacactt aatctcaggc aacgtcgctt 5520 gatgtccgaa gctggcggtg aggtgaaacttacggcaggt gagttcaatc ttctcctcgc 5580 gtttttagag aaaccccgcg acgttctatcgcgcgagcaa cttctcattg ccagtcgagt 5640 acgcgacgag gaggtttatg acaggagtatagatgttctc attttgaggc tgcgccgcaa 5700 acttgaggca gatccgtcaa gccctcaactgataaaaaca gcaagaggtg ccggttattt 5760 ctttgacgcg gacgtgcagg tttcgcacggggggacgatg gcagcctgag ccaattccca 5820 gatccccgag gaatcggcgt gagcggtcgcaaaccatccg gcccggtaca aatcggcgcg 5880 gcgctgggtg atgacctggt ggagaagttgaaggccgcgc aggccgccca gcggcaacgc 5940 atcgaggcag aagcacgccc cggtgaatcgtggcaagcgg ccgctgatcg aatccgcaaa 6000 gaatcccggc aaccgccggc agccggtgcgccgtcgatta ggaagccgcc caagggcgac 6060 gagcaaccag attttttcgt tccgatgctctatgacgtgg gcacccgcga tagtcgcagc 6120 atcatggacg tggccgtttt ccgtctgtcgaagcgtgacc gacgagctgg cgaggtgatc 6180 cgctacgagc ttccagacgg gcacgtagaggtttccgcag ggccggccgg catggccagt 6240 gtgtgggatt acgacctggt actgatggcggtttcccatc taaccgaatc catgaaccga 6300 taccgggaag ggaagggaga caagcccggccgcgtgttcc gtccacacgt tgcggacgta 6360 ctcaagttct gccggcgagc cgatggcggaaagcagaaag acgacctggt agaaacctgc 6420 attcggttaa acaccacgca cgttgccatgcagcgtacga agaaggccaa gaacggccgc 6480 ctggtgacgg tatccgaggg tgaagccttgattagccgct acaagatcgt aaagagcgaa 6540 accgggcggc cggagtacat cgagatcgagctagctgatt ggatgtaccg cgagatcaca 6600 gaaggcaaga acccggacgt gctgacggttcaccccgatt actttttgat cgatcccggc 6660 atcggccgtt ttctctaccg cctggcacgccgcgccgcag gcaaggcaga agccagatgg 6720 ttgttcaaga cgatctacga acgcagtggcagcgccggag agttcaagaa gttctgtttc 6780 accgtgcgca agctgatcgg gtcaaatgacctgccggagt acgatttgaa ggaggaggcg 6840 gggcaggctg gcccgatcct agtcatgcgctaccgcaacc tgatcgaggg cgaagcatcc 6900 gccggttcct aatgtacgga gcagatgctagggcaaattg ccctagcagg ggaaaaaggt 6960 cgaaaaggtc tctttcctgt ggatagcacgtacattggga acccaaagcc gtacattggg 7020 aaccggaacc cgtacattgg gaacccaaagccgtacattg ggaaccggtc acacatgtaa 7080 gtgactgata taaaagagaa aaaaggcgatttttccgcct aaaactcttt aaaacttatt 7140 aaaactctta aaacccgcct ggcctgtgcataactgtctg gccagcgcac agccgaagag 7200 ctgcaaaaag cgcctaccct tcggtcgctgcgctccctac gccccgccgc ttcgcgtcgg 7260 cctatcgcgg ccgctggccg ctcaaaaatggctggcctac ggccaggcaa tctaccaggg 7320 cgcggacaag ccgcgccgtc gccactcgaccgccggcgct gaggtctgcc tcgtgaagaa 7380 ggtgttgctg actcatacca ggcctgaatcgccccatcat ccagccagaa agtgagggag 7440 ccacggttga tgagagcttt gttgtaggtggaccagttgg tgattttgaa cttttgcttt 7500 gccacggaac ggtctgcgtt gtcgggaagatgcgtgatct gatccttcaa ctcagcaaaa 7560 gttcgattta ttcaacaaag ccgccgtcccgtcaagtcag cgtaatgctc tgccagtgtt 7620 acaaccaatt aaccaattct gattagaaaaactcatcgag catcaaatga aactgcaatt 7680 tattcatatc aggattatca ataccatatttttgaaaaag ccgtttctgt aatgaaggag 7740 aaaactcacc gaggcagttc cataggatggcaagatcctg gtatcggtct gcgattccga 7800 ctcgtccaac atcaatacaa cctattaatttcccctcgtc aaaaataagg ttatcaagtg 7860 agaaatcacc atgagtgacg actgaatccggtgagaatgg caaaagctct gcattaatga 7920 atcggccaac gcgcggggag aggcggtttgcgtattgggc gctcttccgc ttcctcgctc 7980 actgactcgc tgcgctcggt cgttcggctgcggcgagcgg tatcagctca ctcaaaggcg 8040 gtaatacggt tatccacaga atcaggggataacgcaggaa agaacatgtg agcaaaaggc 8100 cagcaaaagg ccaggaaccg taaaaaggccgcgttgctgg cgtttttcca taggctccgc 8160 ccccctgacg agcatcacaa aaatcgacgctcaagtcaga ggtggcgaaa cccgacagga 8220 ctataaagat accaggcgtt tccccctggaagctccctcg tgcgctctcc tgttccgacc 8280 ctgccgctta ccggatacct gtccgcctttctcccttcgg gaagcgtggc gctttctcat 8340 agctcacgct gtaggtatct cagttcggtgtaggtcgttc gctccaagct gggctgtgtg 8400 cacgaacccc ccgttcagcc cgaccgctgcgccttatccg gtaactatcg tcttgagtcc 8460 aacccggtaa gacacgactt atcgccactggcagcagcca ctggtaacag gattagcaga 8520 gcgaggtatg taggcggtgc tacagagttcttgaagtggt ggcctaacta cggctacact 8580 agaagaacag tatttggtat ctgcgctctgctgaagccag ttaccttcgg aaaaagagtt 8640 ggtagctctt gatccggcaa acaaaccaccgctggtagcg gtggtttttt tgtttgcaag 8700 cagcagatta cgcgcagaaa aaaaggatctcaagaagatc ctttgatctt ttctacgggg 8760 tctgacgctc agtggaacga aaactcacgttaagggattt tggtcatgag attatcaaaa 8820 aggatcttca cctagatcct tttgatccggaattaattcc tgtggttggc atgcacatac 8880 aaatggacga acggataaac cttttcacgcccttttaaat atccgattat tctaataaac 8940 gctcttttct cttaggttta cccgccaatatatcctgtca aacactgata gtttaaactg 9000 aaggcgggaa acgacaatct gatcatgagcggagaattaa gggagtcacg ttatgacccc 9060 cgccgatgac gcgggacaag ccgttttacgtttggaactg acagaaccgc aacgctgcag 9120 gaattggccg cagcggccat ttaaatcaattgggcgcgta cgtagcacta gtgcgcgatc 9180 gcttaattaa gcggcgcgcc taaagcttctggcagacaaa gtggcagaca tactgtccca 9240 caaatgaaga tggaatctgt aaaagaaaacgcgtgaaata atgcgtctga caaaggttag 9300 gtcggctgcc tttaatcaat accaaagtggtccctaccac gatggaaaaa ctgtgcagtc 9360 ggtttggctt tttctgacga acaaataagattcgtggccg acaggtgggg gtccaccatg 9420 tgaaggcatc ttcagactcc aataatggagcaatgacgta agggcttacg aaataagtaa 9480 gggtagtttg ggaaatgtcc actcacccgtcagtctataa atacttagcc cctccctcat 9540 tgttaaggga gcaag 9555 2 9546 DNAArtificial pNOV2147 2 ggatccccga tcatgcaaaa actcattaac tcagtgcaaaactatgcctg gggcagcaaa 60 acggcgttga ctgaacttta tggtatggaa aatccgtccagccagccgat ggccgagctg 120 tggatgggcg cacatccgaa aagcagttca cgagtgcagaatgccgccgg agatatcgtt 180 tcactgcgtg atgtgattga gagtgataaa tcgactctgctcggagaggc cgttgccaaa 240 cgctttggcg aactgccttt cctgttcaaa gtattatgcgcagcacagcc actctccatt 300 caggttcatc caaacaaaca caattctgaa atcggttttgccaaagaaaa tgccgcaggt 360 atcccgatgg atgccgccga gcgtaactat aaagatcctaaccacaagcc ggagctggtt 420 tttgcgctga cgcctttcct tgcgatgaac gcgtttcgtgaattttccga gattgtctcc 480 ctactccagc cggtcgcagg tgcacatccg gcgattgctcactttttaca acagcctgat 540 gccgaacgtt taagcgaact gttcgccagc ctgttgaatatgcagggtga agaaaaatcc 600 cgcgcgctgg cgattttaaa atcggccctc gatagccagcagggtgaacc gtggcaaacg 660 attcgtttaa tttctgaatt ttacccggaa gacagcggtctgttctcccc gctattgctg 720 aatgtggtga aattgaaccc tggcgaagcg atgttcctgttcgctgaaac accgcacgct 780 tacctgcaag gcgtggcgct ggaagtgatg gcaaactccgataacgtgct gcgtgcgggt 840 ctgacgccta aatacattga tattccggaa ctggttgccaatgtgaaatt cgaagccaaa 900 ccggctaacc agttgttgac ccagccggtg aaacaaggtgcagaactgga cttcccgatt 960 ccagtggatg attttgcctt ctcgctgcat gaccttagtgataaagaaac caccattagc 1020 cagcagagtg ccgccatttt gttctgcgtc gaaggcgatgcaacgttgtg gaaaggttct 1080 cagcagttac agcttaaacc gggtgaatca gcgtttattgccgccaacga atcaccggtg 1140 actgtcaaag gccacggccg tttagcgcgt gtttacaacaagctgtaaga gcttactgaa 1200 aaaattaaca tctcttgcta agctgggagc tctagatccccgaatttccc cgatcgttca 1260 aacatttggc aataaagttt cttaagattg aatcctgttgccggtcttgc gatgattatc 1320 atataatttc tgttgaatta cgttaagcat gtaataattaacatgtaatg catgacgtta 1380 tttatgagat gggtttttat gattagagtc ccgcaattatacatttaata cgcgatagaa 1440 aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcgcggtgtcatc tatgttacta 1500 gatcgggaat tgggtaccat gcccgggcgg ccagcatggccgtatccgca atgtgttatt 1560 aagttgtcta agcgtcaatt tgtttacacc acaatatatcctgccaccag ccagccaaca 1620 gctccccgac cggcagctcg gcacaaaatc accactcgatacaggcagcc catcagaatt 1680 aattctcatg tttgacagct tatcatcgac tgcacggtgcaccaatgctt ctggcgtcag 1740 gcagccatcg gaagctgtgg tatggctgtg caggtcgtaaatcactgcat aattcgtgtc 1800 gctcaaggcg cactcccgtt ctggataatg ttttttgcgccgacatcata acggttctgg 1860 caaatattct gaaatgagct gttgacaatt aatcatccggctcgtataat gtgtggaatt 1920 gtgagcggat aacaatttca cacaggaaac agaccatgagggaagcgttg atcgccgaag 1980 tatcgactca actatcagag gtagttggcg tcatcgagcgccatctcgaa ccgacgttgc 2040 tggccgtaca tttgtacggc tccgcagtgg atggcggcctgaagccacac agtgatattg 2100 atttgctggt tacggtgacc gtaaggcttg atgaaacaacgcggcgagct ttgatcaacg 2160 accttttgga aacttcggct tcccctggag agagcgagattctccgcgct gtagaagtca 2220 ccattgttgt gcacgacgac atcattccgt ggcgttatccagctaagcgc gaactgcaat 2280 ttggagaatg gcagcgcaat gacattcttg caggtatcttcgagccagcc acgatcgaca 2340 ttgatctggc tatcttgctg acaaaagcaa gagaacatagcgttgccttg gtaggtccag 2400 cggcggagga actctttgat ccggttcctg aacaggatctatttgaggcg ctaaatgaaa 2460 ccttaacgct atggaactcg ccgcccgact gggctggcgatgagcgaaat gtagtgctta 2520 cgttgtcccg catttggtac agcgcagtaa ccggcaaaatcgcgccgaag gatgtcgctg 2580 ccgactgggc aatggagcgc ctgccggccc agtatcagcccgtcatactt gaagctaggc 2640 aggcttatct tggacaagaa gatcgcttgg cctcgcgcgcagatcagttg gaagaatttg 2700 ttcactacgt gaaaggcgag atcaccaaag tagtcggcaaataaagctct agtggatctc 2760 cgtacccccg ggggatctgg ctcgcggcgg acgcacgacgccggggcgag accataggcg 2820 atctcctaaa tcaatagtag ctgtaacctc gaagcgtttcacttgtaaca acgattgaga 2880 atttttgtca taaaattgaa atacttggtt cgcatttttgtcatccgcgg tcagccgcaa 2940 ttctgacgaa ctgcccattt agctggagat gattgtacatccttcacgtg aaaatttctc 3000 aagcgctgtg aacaagggtt cagattttag attgaaaggtgagccgttga aacacgttct 3060 tcttgtcgat gacgacgtcg ctatgcggca tcttattattgaatacctta cgatccacgc 3120 cttcaaagtg accgcggtag ccgacagcac ccagttcacaagagtactct cttccgcgac 3180 ggtcgatgtc gtggttgttg atctaaattt aggtcgtgaagatgggctcg agatcgttcg 3240 taatctggcg gcaaagtctg atattccaat cataattatcagtggcgacc gccttgagga 3300 gacggataaa gttgttgcac tcgagctagg agcaagtgattttatcgcta agccgttcag 3360 tatcagagag tttctagcac gcattcgggt tgccttgcgcgtgcgcccca acgttgtccg 3420 ctccaaagac cgacggtctt tttgttttac tgactggacacttaatctca ggcaacgtcg 3480 cttgatgtcc gaagctggcg gtgaggtgaa acttacggcaggtgagttca atcttctcct 3540 cgcgttttta gagaaacccc gcgacgttct atcgcgcgagcaacttctca ttgccagtcg 3600 agtacgcgac gaggaggttt atgacaggag tatagatgttctcattttga ggctgcgccg 3660 caaacttgag gcagatccgt caagccctca actgataaaaacagcaagag gtgccggtta 3720 tttctttgac gcggacgtgc aggtttcgca cggggggacgatggcagcct gagccaattc 3780 ccagatcccc gaggaatcgg cgtgagcggt cgcaaaccatccggcccggt acaaatcggc 3840 gcggcgctgg gtgatgacct ggtggagaag ttgaaggccgcgcaggccgc ccagcggcaa 3900 cgcatcgagg cagaagcacg ccccggtgaa tcgtggcaagcggccgctga tcgaatccgc 3960 aaagaatccc ggcaaccgcc ggcagccggt gcgccgtcgattaggaagcc gcccaagggc 4020 gacgagcaac cagatttttt cgttccgatg ctctatgacgtgggcacccg cgatagtcgc 4080 agcatcatgg acgtggccgt tttccgtctg tcgaagcgtgaccgacgagc tggcgaggtg 4140 atccgctacg agcttccaga cgggcacgta gaggtttccgcagggccggc cggcatggcc 4200 agtgtgtggg attacgacct ggtactgatg gcggtttcccatctaaccga atccatgaac 4260 cgataccggg aagggaaggg agacaagccc ggccgcgtgttccgtccaca cgttgcggac 4320 gtactcaagt tctgccggcg agccgatggc ggaaagcagaaagacgacct ggtagaaacc 4380 tgcattcggt taaacaccac gcacgttgcc atgcagcgtacgaagaaggc caagaacggc 4440 cgcctggtga cggtatccga gggtgaagcc ttgattagccgctacaagat cgtaaagagc 4500 gaaaccgggc ggccggagta catcgagatc gagctagctgattggatgta ccgcgagatc 4560 acagaaggca agaacccgga cgtgctgacg gttcaccccgattacttttt gatcgatccc 4620 ggcatcggcc gttttctcta ccgcctggca cgccgcgccgcaggcaaggc agaagccaga 4680 tggttgttca agacgatcta cgaacgcagt ggcagcgccggagagttcaa gaagttctgt 4740 ttcaccgtgc gcaagctgat cgggtcaaat gacctgccggagtacgattt gaaggaggag 4800 gcggggcagg ctggcccgat cctagtcatg cgctaccgcaacctgatcga gggcgaagca 4860 tccgccggtt cctaatgtac ggagcagatg ctagggcaaattgccctagc aggggaaaaa 4920 ggtcgaaaag gtctctttcc tgtggatagc acgtacattgggaacccaaa gccgtacatt 4980 gggaaccgga acccgtacat tgggaaccca aagccgtacattgggaaccg gtcacacatg 5040 taagtgactg atataaaaga gaaaaaaggc gatttttccgcctaaaactc tttaaaactt 5100 attaaaactc ttaaaacccg cctggcctgt gcataactgtctggccagcg cacagccgaa 5160 gagctgcaaa aagcgcctac ccttcggtcg ctgcgctccctacgccccgc cgcttcgcgt 5220 cggcctatcg cggccgctgg ccgctcaaaa atggctggcctacggccagg caatctacca 5280 gggcgcggac aagccgcgcc gtcgccactc gaccgccggcgctgaggtct gcctcgtgaa 5340 gaaggtgttg ctgactcata ccaggcctga atcgccccatcatccagcca gaaagtgagg 5400 gagccacggt tgatgagagc tttgttgtag gtggaccagttggtgatttt gaacttttgc 5460 tttgccacgg aacggtctgc gttgtcggga agatgcgtgatctgatcctt caactcagca 5520 aaagttcgat ttattcaaca aagccgccgt cccgtcaagtcagcgtaatg ctctgccagt 5580 gttacaacca attaaccaat tctgattaga aaaactcatcgagcatcaaa tgaaactgca 5640 atttattcat atcaggatta tcaataccat atttttgaaaaagccgtttc tgtaatgaag 5700 gagaaaactc accgaggcag ttccatagga tggcaagatcctggtatcgg tctgcgattc 5760 cgactcgtcc aacatcaata caacctatta atttcccctcgtcaaaaata aggttatcaa 5820 gtgagaaatc accatgagtg acgactgaat ccggtgagaatggcaaaagc tctgcattaa 5880 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattgggcgctcttc cgcttcctcg 5940 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgagcggtatcagc tcactcaaag 6000 gcggtaatac ggttatccac agaatcaggg gataacgcaggaaagaacat gtgagcaaaa 6060 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgctggcgttttt ccataggctc 6120 cgcccccctg acgagcatca caaaaatcga cgctcaagtcagaggtggcg aaacccgaca 6180 ggactataaa gataccaggc gtttccccct ggaagctccctcgtgcgctc tcctgttccg 6240 accctgccgc ttaccggata cctgtccgcc tttctcccttcgggaagcgt ggcgctttct 6300 catagctcac gctgtaggta tctcagttcg gtgtaggtcgttcgctccaa gctgggctgt 6360 gtgcacgaac cccccgttca gcccgaccgc tgcgccttatccggtaacta tcgtcttgag 6420 tccaacccgg taagacacga cttatcgcca ctggcagcagccactggtaa caggattagc 6480 agagcgaggt atgtaggcgg tgctacagag ttcttgaagtggtggcctaa ctacggctac 6540 actagaagaa cagtatttgg tatctgcgct ctgctgaagccagttacctt cggaaaaaga 6600 gttggtagct cttgatccgg caaacaaacc accgctggtagcggtggttt ttttgtttgc 6660 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaagatcctttgat cttttctacg 6720 gggtctgacg ctcagtggaa cgaaaactca cgttaagggattttggtcat gagattatca 6780 aaaaggatct tcacctagat ccttttgatc cggaattaattcctgtggtt ggcatgcaca 6840 tacaaatgga cgaacggata aaccttttca cgcccttttaaatatccgat tattctaata 6900 aacgctcttt tctcttaggt ttacccgcca atatatcctgtcaaacactg atagtttaaa 6960 ctgaaggcgg gaaacgacaa tctgatcatg agcggagaattaagggagtc acgttatgac 7020 ccccgccgat gacgcgggac aagccgtttt acgtttggaactgacagaac cgcaacgctg 7080 caggaattgg ccgcagcggc catttaaatc aattgggcgcgtacgtagca ctagtgcgcg 7140 atcgcttaat taagcggcgc gcctaaagct tctggcagacaaagtggcag acatactgtc 7200 ccacaaatga agatggaatc tgtaaaagaa aacgcgtgaaataatgcgtc tgacaaaggt 7260 taggtcggct gcctttaatc aataccaaag tggtccctaccacgatggaa aaactgtgca 7320 gtcggtttgg ctttttctga cgaacaaata agattcgtggccgacaggtg ggggtccacc 7380 atgtgaaggc atcttcagac tccaataatg gagcaatgacgtaagggctt acgaaataag 7440 taagggtagt ttgggaaatg tccactcacc cgtcagtctataaatactta gcccctccct 7500 cattgttaag ggagcaagga tccaccggtc gccaccatggccctgtccaa caagttcatc 7560 ggcgacgaca tgaagatgac ctaccacatg gacggctgcgtgaacggcca ctacttcacc 7620 gtgaagggcg agggcagcgg caagccctac gagggcacccagacctccac cttcaaggtg 7680 accatggcca acggcggccc cctggccttc tccttcgacatcctgtccac cgtgttcatg 7740 tacggcaacc gctgcttcac cgcctacccc accagcatgcccgactactt caagcaggcc 7800 ttccccgacg gcatgtccta cgagagaacc ttcacctacgaggacggcgg cgtggccacc 7860 gccagctggg agatcagcct gaagggcaac tgcttcgagcacaagtccac cttccacggc 7920 gtgaacttcc ccgccgacgg ccccgtgatg gccaagaagaccaccggctg ggacccctcc 7980 ttcgagaaga tgaccgtgtg cgacggcatc ttgaagggcgacgtgaccgc cttcctgatg 8040 ctgcagggcg gcggcaacta cagatgccag ttccacacctcctacaagac caagaagccc 8100 gtgaccatgc cccccaacca cgtggtggag caccgcatcgccagaaccga cctggacaag 8160 ggcggcaaca gcgtgcagct gaccgagcac gccgtggcccacatcacctc cgtggtgccc 8220 ttctgagagc tctagatccc cgaatttccc cgatcgttcaaacatttggc aataaagttt 8280 cttaagattg aatcctgttg ccggtcttgc gatgattatcatataatttc tgttgaatta 8340 cgttaagcat gtaataatta acatgtaatg catgacgttatttatgagat gggtttttat 8400 gattagagtc ccgcaattat acatttaata cgcgatagaaaacaaaatat agcgcgcaaa 8460 ctaggataaa ttatcgcgcg cggtgtcatc tatgttactagatcgggaat tgggtaccga 8520 attcactggc cgtcgtttta caacgtcgtg actgggaaaaccctggcgtt acccaactta 8580 atcgccttgc agcacatccc cctttcgcca gctggcgtaatagcgaagag gcccgcaccg 8640 atcgcccttc ccaacagttg cgcagcctga atggcgaatggcgcctgatg cggtattttc 8700 tccttacgca tctgtgcggt atttcacacc gcatatggtgcactctcagt acaatctgct 8760 ctgatgccgc atagttaagc cagccccgac acccgccaacacccgctgac gcgccctgac 8820 gggcttgtct gctcccggca tccgcttaca gacaagctgtgaccgtctcc gggagctgca 8880 tgtgtcagag gttttcaccg tcatcaccga aacgcgcgagacgaaagggc ctcgtgatac 8940 gcctattttt ataggttaat gtcatgataa taatggtttcttagacgtca ggtggcactt 9000 ttcggggaaa tgtgcgcgga acccctattt gtttatttttctaaatacat tcaaatatgt 9060 atccgctcat gagacaataa ccctgataaa tgcttcaatggcgcgccggt accagcttgc 9120 atgcctgcag gtcgactcta gaggatcctg gcagacaaagtggcagacat actgtcccac 9180 aaatgaagat ggaatctgta aaagaaaacg cgtgaaataatgcgtctgac aaaggttagg 9240 tcggctgcct ttaatcaata ccaaagtggt ccctaccacgatggaaaaac tgtgcagtcg 9300 gtttggcttt ttctgacgaa caaataagat tcgtggccgacaggtggggg tccaccatgt 9360 gaaggcatct tcagactcca ataatggagc aatgacgtaagggcttacga aataagtaag 9420 ggtagtttgg gaaatgtcca ctcacccgtc agtctataaatacttagccc ctccctcatt 9480 gttaagggag caaaatctca gagagatagt cctagagagagaaagagagc aagtagccta 9540 gaagta 9546 3 10604 DNA Artificial pBSC112343 cgcgcctaaa gcttgcatgc ctgcaggtcg actctagagg atcctggcag acaaagtggc 60agacatactg tcccacaaat gaagatggaa tctgtaaaag aaaacgcgtg aaataatgcg 120tctgacaaag gttaggtcgg ctgcctttaa tcaataccaa agtggtccct accacgatgg 180aaaaactgtg cagtcggttt ggctttttct gacgaacaaa taagattcgt ggccgacagg 240tgggggtcca ccatgtgaag gcatcttcag actccaataa tggagcaatg acgtaagggc 300ttacgaaata agtaagggta gtttgggaaa tgtccactca cccgtcagtc tataaatact 360tagcccctcc ctcattgtta agggagcaaa atctcagaga gatagtccta gagagagaaa 420gagagcaagt agcctagaag taggatcccc gatcatgcaa aaactcatta actcagtgca 480aaactatgcc tggggcagca aaacggcgtt gactgaactt tatggtatgg aaaatccgtc 540cagccagccg atggccgagc tgtggatggg cgcacatccg aaaagcagtt cacgagtgca 600gaatgccgcc ggagatatcg tttcactgcg tgatgtgatt gagagtgata aatcgactct 660gctcggagag gccgttgcca aacgctttgg cgaactgcct ttcctgttca aagtattatg 720cgcagcacag ccactctcca ttcaggttca tccaaacaaa cacaattctg aaatcggttt 780tgccaaagaa aatgccgcag gtatcccgat ggatgccgcc gagcgtaact ataaagatcc 840taaccacaag ccggagctgg tttttgcgct gacgcctttc cttgcgatga acgcgtttcg 900tgaattttcc gagattgtct ccctactcca gccggtcgca ggtgcacatc cggcgattgc 960tcacttttta caacagcctg atgccgaacg tttaagcgaa ctgttcgcca gcctgttgaa 1020tatgcagggt gaagaaaaat cccgcgcgct ggcgatttta aaatcggccc tcgatagcca 1080gcagggtgaa ccgtggcaaa cgattcgttt aatttctgaa ttttacccgg aagacagcgg 1140tctgttctcc ccgctattgc tgaatgtggt gaaattgaac cctggcgaag cgatgttcct 1200gttcgctgaa acaccgcacg cttacctgca aggcgtggcg ctggaagtga tggcaaactc 1260cgataacgtg ctgcgtgcgg gtctgacgcc taaatacatt gatattccgg aactggttgc 1320caatgtgaaa ttcgaagcca aaccggctaa ccagttgttg acccagccgg tgaaacaagg 1380tgcagaactg gacttcccga ttccagtgga tgattttgcc ttctcgctgc atgaccttag 1440tgataaagaa accaccatta gccagcagag tgccgccatt ttgttctgcg tcgaaggcga 1500tgcaacgttg tggaaaggtt ctcagcagtt acagcttaaa ccgggtgaat cagcgtttat 1560tgccgccaac gaatcaccgg tgactgtcaa aggccacggc cgtttagcgc gtgtttacaa 1620caagctgtaa gagcttactg aaaaaattaa catctcttgc taagctggga gctctagatc 1680cccgaatttc cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt 1740tgccggtctt gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat 1800taacatgtaa tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt 1860atacatttaa tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg 1920cgcggtgtca tctatgttac tagatcggga attgggtacc atgcccgggc ggccagcatg 1980gccgtatccg caatgtgtta ttaagttgtc taagcgtcaa tttgtttaca ccacaatata 2040tcctgccacc agccagccaa cagctccccg accggcagct cggcacaaaa tcaccactcg 2100atacaggcag cccatcagaa ttaattctca tgtttgacag cttatcatcg actgcacggt 2160gcaccaatgc ttctggcgtc aggcagccat cggaagctgt ggtatggctg tgcaggtcgt 2220aaatcactgc ataattcgtg tcgctcaagg cgcactcccg ttctggataa tgttttttgc 2280gccgacatca taacggttct ggcaaatatt ctgaaatgag ctgttgacaa ttaatcatcc 2340ggctcgtata atgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagaccatg 2400agggaagcgt tgatcgccga agtatcgact caactatcag aggtagttgg cgtcatcgag 2460cgccatctcg aaccgacgtt gctggccgta catttgtacg gctccgcagt ggatggcggc 2520ctgaagccac acagtgatat tgatttgctg gttacggtga ccgtaaggct tgatgaaaca 2580acgcggcgag ctttgatcaa cgaccttttg gaaacttcgg cttcccctgg agagagcgag 2640attctccgcg ctgtagaagt caccattgtt gtgcacgacg acatcattcc gtggcgttat 2700ccagctaagc gcgaactgca atttggagaa tggcagcgca atgacattct tgcaggtatc 2760ttcgagccag ccacgatcga cattgatctg gctatcttgc tgacaaaagc aagagaacat 2820agcgttgcct tggtaggtcc agcggcggag gaactctttg atccggttcc tgaacaggat 2880ctatttgagg cgctaaatga aaccttaacg ctatggaact cgccgcccga ctgggctggc 2940gatgagcgaa atgtagtgct tacgttgtcc cgcatttggt acagcgcagt aaccggcaaa 3000atcgcgccga aggatgtcgc tgccgactgg gcaatggagc gcctgccggc ccagtatcag 3060cccgtcatac ttgaagctag gcaggcttat cttggacaag aagatcgctt ggcctcgcgc 3120gcagatcagt tggaagaatt tgttcactac gtgaaaggcg agatcaccaa agtagtcggc 3180aaataaagct ctagtggatc tccgtacccc cgggggatct ggctcgcggc ggacgcacga 3240cgccggggcg agaccatagg cgatctccta aatcaatagt agctgtaacc tcgaagcgtt 3300tcacttgtaa caacgattga gaatttttgt cataaaattg aaatacttgg ttcgcatttt 3360tgtcatccgc ggtcagccgc aattctgacg aactgcccat ttagctggag atgattgtac 3420atccttcacg tgaaaatttc tcaagcgctg tgaacaaggg ttcagatttt agattgaaag 3480gtgagccgtt gaaacacgtt cttcttgtcg atgacgacgt cgctatgcgg catcttatta 3540ttgaatacct tacgatccac gccttcaaag tgaccgcggt agccgacagc acccagttca 3600caagagtact ctcttccgcg acggtcgatg tcgtggttgt tgatctaaat ttaggtcgtg 3660aagatgggct cgagatcgtt cgtaatctgg cggcaaagtc tgatattcca atcataatta 3720tcagtggcga ccgccttgag gagacggata aagttgttgc actcgagcta ggagcaagtg 3780attttatcgc taagccgttc agtatcagag agtttctagc acgcattcgg gttgccttgc 3840gcgtgcgccc caacgttgtc cgctccaaag accgacggtc tttttgtttt actgactgga 3900cacttaatct caggcaacgt cgcttgatgt ccgaagctgg cggtgaggtg aaacttacgg 3960caggtgagtt caatcttctc ctcgcgtttt tagagaaacc ccgcgacgtt ctatcgcgcg 4020agcaacttct cattgccagt cgagtacgcg acgaggaggt ttatgacagg agtatagatg 4080ttctcatttt gaggctgcgc cgcaaacttg aggcagatcc gtcaagccct caactgataa 4140aaacagcaag aggtgccggt tatttctttg acgcggacgt gcaggtttcg cacgggggga 4200cgatggcagc ctgagccaat tcccagatcc ccgaggaatc ggcgtgagcg gtcgcaaacc 4260atccggcccg gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc 4320cgcgcaggcc gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca 4380agcggccgct gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc 4440gattaggaag ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga 4500cgtgggcacc cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg 4560tgaccgacga gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc 4620cgcagggccg gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc 4680ccatctaacc gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt 4740gttccgtcca cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca 4800gaaagacgac ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg 4860tacgaagaag gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag 4920ccgctacaag atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc 4980tgattggatg taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc 5040cgattacttt ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc 5100cgcaggcaag gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc 5160cggagagttc aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc 5220ggagtacgat ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg 5280caacctgatc gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca 5340aattgcccta gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat 5400tgggaaccca aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta 5460cattgggaac cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc 5520cgcctaaaac tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact 5580gtctggccag cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc 5640cctacgcccc gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg 5700cctacggcca ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg 5760gcgctgaggt ctgcctcgtg aagaaggtgt tgctgactca taccaggcct gaatcgcccc 5820atcatccagc cagaaagtga gggagccacg gttgatgaga gctttgttgt aggtggacca 5880gttggtgatt ttgaactttt gctttgccac ggaacggtct gcgttgtcgg gaagatgcgt 5940gatctgatcc ttcaactcag caaaagttcg atttattcaa caaagccgcc gtcccgtcaa 6000gtcagcgtaa tgctctgcca gtgttacaac caattaacca attctgatta gaaaaactca 6060tcgagcatca aatgaaactg caatttattc atatcaggat tatcaatacc atatttttga 6120aaaagccgtt tctgtaatga aggagaaaac tcaccgaggc agttccatag gatggcaaga 6180tcctggtatc ggtctgcgat tccgactcgt ccaacatcaa tacaacctat taatttcccc 6240tcgtcaaaaa taaggttatc aagtgagaaa tcaccatgag tgacgactga atccggtgag 6300aatggcaaaa gctctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat 6360tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 6420agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 6480aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 6540gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 6600tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 6660cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 6720ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 6780cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 6840atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 6900agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 6960gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 7020gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 7080tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 7140agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 7200gattttggtc atgagattat caaaaaggat cttcacctag atccttttga tccggaatta 7260attcctgtgg ttggcatgca catacaaatg gacgaacgga taaacctttt cacgcccttt 7320taaatatccg attattctaa taaacgctct tttctcttag gtttacccgc caatatatcc 7380tgtcaaacac tgatagttta aactgaaggc gggaaacgac aatctgatca tgagcggaga 7440attaagggag tcacgttatg acccccgccg atgacgcggg acaagccgtt ttacgtttgg 7500aactgacaga accgcaacgc tgcaggaatt ggccgcagcg gccatttaaa tcaattgggc 7560gcgtacgtag cactagtgcg cgatcgctta attaagcggc gcgcctgcag gcggccgcac 7620aattattata tcaaaatggc aaaaacattt aatacgtatt atttaagaaa aaaatatgta 7680ataatatatt tatattttaa tatctattct tatgtatttt ttaaaaatct attatatatt 7740gatcaactaa aatattttta tatctacact tattttgcat ttttatcaat tttcttgcgt 7800tttttggcat atttaataat gactattctt taataatcga tcattattct tacatggtac 7860atattgttgg aaccatatga agtgtccatt gcatttgact atgtggatag tgttttgatc 7920caggcctcca tttgccgctt attaattaat ttggtaacag tccgtactaa tcagttactt 7980atccttcctc catcataatt aatcttggta gtctcgaatg ccacaacact gactagtctc 8040ttggatcata agaaaaagcc aaggaacaaa agaagacaaa acacaatggg agtatccttt 8100gcatagcaat gtctaagttc ataaaattca aacaaaaacg caatcacaca cagtggacat 8160cacttatcca ctagctgatc aggatcgccg cgtcaagaaa aaaaaactgg accccaaaag 8220ccatgcacaa caacacgtac tcacaaaggt gtcaatcgag cagcccaaaa cattcaccaa 8280ctcaacccat catgagccca cacatttgtt gtttctaacc caacctcaaa ctcgtattct 8340cttccgccac ctcatttttg tttatttcaa cacccgtcaa actgcatgcc accccgtggc 8400caaatgtcca tgcatgttaa caagacctat gactataaat atctgcaatc tcggcccagg 8460ttttcatcat caagaaccag ttcaatatcc tagtacaccg tattaaagaa tttaagatat 8520actccaccgg atccaccatg gccaagctag ttttttccct ttgttttctg cttttcagtg 8580gctgctgctt cgctgagatt ttcggcaaga ccttccgcga gggccgcttc gtgctcaagg 8640agaagaactt caccgtggag ttcgccgtgg agaagatcca cctcggctgg aagatatcgg 8700gccgcgtgaa gggctcgccg ggccgcctcg aggtgctccg caccaaggcc ccggagaagg 8760tgctcgtgaa caactggcag tcctggggcc cgtgccgcgt ggtggacgcc ttctccttca 8820agccgccgga gatcgacccg aactggcgct acaccgcatc cgtggtgccg gacgtgctcg 8880agcgcaacct gcagtccgac tacttcgtgg ccgaggaggg caaggtgtac ggcttcctct 8940cctccaagat cgcccacccg ttcttcgcgg tggaggacgg cgagctggtg gcctacctcg 9000agtacttcga cgtggagttc gacgacttcg tgccgctgga gccgctcgtg gtgctcgagg 9060acccgaacac cccgctcctc ctcgagaagt acgccgagct ggtgggcatg gagaacaacg 9120cccgggtgcc gaagcacacg ccgaccggct ggtgctcctg gtatcactac ttcctcgacc 9180tcacctggga ggagaccctc aagaacctca agctcgccaa gaacttcccg ttcgaggtgt 9240tccagatcga cgacgcctac gagaaggaca tcggcgactg gctcgtgacc cgcggcgact 9300tcccgtccgt ggaggagatg gccaaggtga tcgccgagaa cggcttcatc cccggcatct 9360ggaccgcccc gttctccgtg tccgagacta gtgacgtgtt caacgagcac ccggactggg 9420tggtgaagga gaacggcgag ccgaagatgg cctaccgcaa ctggaacaag aagatttacg 9480ccctcgacct ctccaaggac gaggtgctca actggctctt cgacctcttc tcctccctcc 9540gcaagatggg ctaccgctac ttcaagatcg acttcctctt cgcgggcgcc gtgccggggg 9600agcgcaagaa gaacatcacc ccgatccagg ccttccgcaa gggcatcgag accatccgca 9660aggccgtggg ggaggactcc ttcatcctcg gctgcggctc ccccctcctc ccggccgtgg 9720gctgcgtgga tggcatgcgc atcggcccgg acaccgcccc gttctgggga gagcacatcg 9780aggacaacgg cgccccggcg gcccgctggg ccctccgcaa cgccatcacc cgctacttca 9840tgcacgaccg cttctggctc aacgacccgg actgcctcat cctccgcgag gagaagaccg 9900acctcaccca gaaggagaag gagctgtact cctacacctg cggcgttcta gacaacatga 9960tcatcgagtc cgacgacctc tccctcgtgc gcgaccacgg caagaaggtg ctcaaggaga 10020ccctcgagct gctcgggggc aggccgcgcg tgcagaacat catgtccgag gacctccgct 10080acgagatcgt gtcctcgggc accctctccg gcaacgtgaa gatcgtggtg gacctcaact 10140cccgcgagta ccacctcgag aaggagggca agtcctccct caagaagcgc gtggtgaagc 10200gggaggacgg caggaacttc tacttctacg aggagggcga gcgcgagtga aagcttgacg 10260tcactagtgc gatcgcgcta gccatggccg gcctaggcgc ccgggagatc cccgaatttc 10320cccgatcgtt caaacatttg gcaataaagt ttcttaagat tgaatcctgt tgccggtctt 10380gcgatgatta tcatataatt tctgttgaat tacgttaagc atgtaataat taacatgtaa 10440tgcatgacgt tatttatgag atgggttttt atgattagag tcccgcaatt atacatttaa 10500tacgcgatag aaaacaaaat atagcgcgca aactaggata aattatcgcg cgcggtgtca 10560tctatgttac tagatcggga attcctcgag tctagacctg cagg 10604 4 8757 DNAArtificial pBSC11369 4 aagcttctgg cagacaaagt ggcagacata ctgtcccacaaatgaagatg gaatctgtaa 60 aagaaaacgc gtgaaataat gcgtctgaca aaggttaggtcggctgcctt taatcaatac 120 caaagtggtc cctaccacga tggaaaaact gtgcagtcggtttggctttt tctgacgaac 180 aaataagatt cgtggccgac aggtgggggt ccaccatgtgaaggcatctt cagactccaa 240 taatggagca atgacgtaag ggcttacgaa ataagtaagggtagtttggg aaatgtccac 300 tcacccgtca gtctataaat acttagcccc tccctcattgttaagggagc aaggatccac 360 cggtcgccac catggcccag tccaagcacg gcctgaccaaggagatgacc atgaagtacc 420 gcatggaggg ctgcgtggac ggccacaagt tcgtgatcaccggcgagggc atcggctacc 480 ccttcaaggg caagcaggcc atcaacctgt gcgtggtggagggcggcccc ttgcccttcg 540 ccgaggacat cttgtccgcc gccttcatgt acggcaaccgcgtgttcacc gagtaccccc 600 aggacatcgt cgactacttc aagaactcct gccccgccggctacacctgg gaccgctcct 660 tcctgttcga ggacggcgcc gtgtgcatct gcaacgccgacatcaccgtg agcgtggagg 720 agaactgcat gtaccacgag tccaagttct acggcgtgaacttccccgcc gacggccccg 780 tgatgaagaa gatgaccgac aactgggagc cctcctgcgagaagatcatc cccgtgccca 840 agcagggcat cttgaagggc gacgtgagca tgtacctgctgctgaaggac ggtggccgct 900 tgcgctgcca gttcgacacc gtgtacaagg ccaagtccgtgccccgcaag atgcccgact 960 ggcacttcat ccagcacaag ctgacccgcg aggaccgcagcgacgccaag aaccagaagt 1020 ggcacctgac cgagcacgcc atcgcctccg gctccgccttgccctgctct agatcccgaa 1080 tttccccgat cgttcaaaca tttggcaata aagtttcttaagattgaatc ctgttgccgg 1140 tcttgcgatg attatcatat aatttctgtt gaattacgttaagcatgtaa taattaacat 1200 gtaatgcatg acgttattta tgagatgggt ttttatgattagagtcccgc aattatacat 1260 ttaatacgcg atagaaaaca aaatatagcg cgcaaactaggataaattat cgcgcgcggt 1320 gtcatctatg ttactagatc gggaattggg gaaatttaccggtgccgaat ttccccgatc 1380 cagcttctgg cagacaaagt ggcagacata ctgtcccacaaatgaagatg gaatctgtaa 1440 aagaaaacgc gtgaaataat gcgtctgaca aaggttaggtcggctgcctt taatcaatac 1500 caaagtggtc cctaccacga tggaaaaact gtgcagtcggtttggctttt tctgacgaac 1560 aaataagatt cgtggccgac aggtgggggt ccaccatgtgaaggcatctt cagactccaa 1620 taatggagca atgacgtaag ggcttacgaa ataagtaagggtagtttggg aaatgtccac 1680 tcacccgtca gtctataaat acttagcccc tccctcattgttaagggagc aaggatccat 1740 gaaaaagcct gaactcaccg cgacgtctgt cgagaagtttctgatcgaaa agttcgacag 1800 cgtctccgac ctgatgcagc tctcggaggg cgaagaatctcgtgctttca gcttcgatgt 1860 aggagggcgt ggatatgtcc tgcgggtaaa tagctgcgccgatggtttct acaaagatcg 1920 ttatgtttat cggcactttg catcggccgc gctcccgattccggaagtgc ttgacattgg 1980 ggaattcagc gagagcctga cctattgcat ctcccgccgtgcacagggtg tcacgttgca 2040 agacctgcct gaaaccgaac tgcccgctgt tctgcagccggtcgcggagg ccatggatgc 2100 gatcgctgcg gccgatctta gccagacgag cgggttcggcccattcggac cgcaaggaat 2160 cggtcaatac actacatggc gtgatttcat atgcgcgattgctgatcccc atgtgtatca 2220 ctggcaaact gtgatggacg acaccgtcag tgcgtccgtcgcgcaggctc tcgatgagct 2280 gatgctttgg gccgaggact gccccgaagt ccggcacctcgtgcacgcgg atttcggctc 2340 caacaatgtc ctgacggaca atggccgcat aacagcggtcattgactgga gcgaggcgat 2400 gttcggggat tcccaatacg aggtcgccaa catcttcttctggaggccgt ggttggcttg 2460 tatggagcag cagacgcgct acttcgagcg gaggcatccggagcttgcag gatcgccgcg 2520 gctccgggcg tatatgctcc gcattggtct tgaccaactctatcagagct tggttgacgg 2580 caatttcgat gatgcagctt gggcgcaggg tcgatgcgacgcaatcgtcc gatccggagc 2640 cgggactgtc gggcgtacac aaatcgcccg cagaagcgcggccgtctgga ccgatggctg 2700 tgtagaagta ctcgccgata gtggaaaccg acgccccagcactcgtccga gggcaaagga 2760 atagggatcc cccgaatttc cccgatcgtt caaacatttggcaataaagt ttcttaagat 2820 tgaatcctgt tgccggtctt gcgatgatta tcatataatttctgttgaat tacgttaagc 2880 atgtaataat taacatgtaa tgcatgacgt tatttatgagatgggttttt atgattagag 2940 tcccgcaatt atacatttaa tacgcgatag aaaacaaaatatagcgcgca aactaggata 3000 aattatcgcg cgcggtgtca tctatgttac tagatcgggaattagcggcc cgaattcact 3060 ggccgtcgtt ttacaacgtc gtgactggga aaaccctggcgttacccaac ttaatcgcct 3120 tgcagcacat ccccctttcg ccaggggcgg ccagcatggccgtatccgca atgtgttatt 3180 aagttgtcta agcgtcaatt tgtttacacc acaatatatcctgccaccag ccagccaaca 3240 gctccccgac cggcagctcg gcacaaaatc accactcgatacaggcagcc catcagaatt 3300 aattctcatg tttgacagct tatcatcgac tgcacggtgcaccaatgctt ctggcgtcag 3360 gcagccatcg gaagctgtgg tatggctgtg caggtcgtaaatcactgcat aattcgtgtc 3420 gctcaaggcg cactcccgtt ctggataatg ttttttgcgccgacatcata acggttctgg 3480 caaatattct gaaatgagct gttgacaatt aatcatccggctcgtataat gtgtggaatt 3540 gtgagcggat aacaatttca cacaggaaac agaccatgagggaagcgttg atcgccgaag 3600 tatcgactca actatcagag gtagttggcg tcatcgagcgccatctcgaa ccgacgttgc 3660 tggccgtaca tttgtacggc tccgcagtgg atggcggcctgaagccacac agtgatattg 3720 atttgctggt tacggtgacc gtaaggcttg atgaaacaacgcggcgagct ttgatcaacg 3780 accttttgga aacttcggct tcccctggag agagcgagattctccgcgct gtagaagtca 3840 ccattgttgt gcacgacgac atcattccgt ggcgttatccagctaagcgc gaactgcaat 3900 ttggagaatg gcagcgcaat gacattcttg caggtatcttcgagccagcc acgatcgaca 3960 ttgatctggc tatcttgctg acaaaagcaa gagaacatagcgttgccttg gtaggtccag 4020 cggcggagga actctttgat ccggttcctg aacaggatctatttgaggcg ctaaatgaaa 4080 ccttaacgct atggaactcg ccgcccgact gggctggcgatgagcgaaat gtagtgctta 4140 cgttgtcccg catttggtac agcgcagtaa ccggcaaaatcgcgccgaag gatgtcgctg 4200 ccgactgggc aatggagcgc ctgccggccc agtatcagcccgtcatactt gaagctaggc 4260 aggcttatct tggacaagaa gatcgcttgg cctcgcgcgcagatcagttg gaagaatttg 4320 ttcactacgt gaaaggcgag atcaccaaag tagtcggcaaataaagctct agtggatctc 4380 cgtacccggg gatctggctc gcggcggacg cacgacgccggggcgagacc ataggcgatc 4440 tcctaaatca atagtagctg taacctcgaa gcgtttcacttgtaacaacg attgagaatt 4500 tttgtcataa aattgaaata cttggttcgc atttttgtcatccgcggtca gccgcaattc 4560 tgacgaactg cccatttagc tggagatgat tgtacatccttcacgtgaaa atttctcaag 4620 cgctgtgaac aagggttcag attttagatt gaaaggtgagccgttgaaac acgttcttct 4680 tgtcgatgac gacgtcgcta tgcggcatct tattattgaataccttacga tccacgcctt 4740 caaagtgacc gcggtagccg acagcaccca gttcacaagagtactctctt ccgcgacggt 4800 cgatgtcgtg gttgttgatc tagatttagg tcgtgaagatgggctcgaga tcgttcgtaa 4860 tctggcggca aagtctgata ttccaatcat aattatcagtggcgaccgcc ttgaggagac 4920 ggataaagtt gttgcactcg agctaggagc aagtgattttatcgctaagc cgttcagtat 4980 cagagagttt ctagcacgca ttcgggttgc cttgcgcgtgcgccccaacg ttgtccgctc 5040 caaagaccga cggtcttttt gttttactga ctggacacttaatctcaggc aacgtcgctt 5100 gatgtccgaa gctggcggtg aggtgaaact tacggcaggtgagttcaatc ttctcctcgc 5160 gtttttagag aaaccccgcg acgttctatc gcgcgagcaacttctcattg ccagtcgagt 5220 acgcgacgag gaggtttatg acaggagtat agatgttctcattttgaggc tgcgccgcaa 5280 acttgaggca gatccgtcaa gccctcaact gataaaaacagcaagaggtg ccggttattt 5340 ctttgacgcg gacgtgcagg tttcgcacgg ggggacgatggcagcctgag ccaattccca 5400 gatccccgag gaatcggcgt gagcggtcgc aaaccatccggcccggtaca aatcggcgcg 5460 gcgctgggtg atgacctggt ggagaagttg aaggccgcgcaggccgccca gcggcaacgc 5520 atcgaggcag aagcacgccc cggtgaatcg tggcaagcggccgctgatcg aatccgcaaa 5580 gaatcccggc aaccgccggc agccggtgcg ccgtcgattaggaagccgcc caagggcgac 5640 gagcaaccag attttttcgt tccgatgctc tatgacgtgggcacccgcga tagtcgcagc 5700 atcatggacg tggccgtttt ccgtctgtcg aagcgtgaccgacgagctgg cgaggtgatc 5760 cgctacgagc ttccagacgg gcacgtagag gtttccgcagggccggccgg catggccagt 5820 gtgtgggatt acgacctggt actgatggcg gtttcccatctaaccgaatc catgaaccga 5880 taccgggaag ggaagggaga caagcccggc cgcgtgttccgtccacacgt tgcggacgta 5940 ctcaagttct gccggcgagc cgatggcgga aagcagaaagacgacctggt agaaacctgc 6000 attcggttaa acaccacgca cgttgccatg cagcgtacgaagaaggccaa gaacggccgc 6060 ctggtgacgg tatccgaggg tgaagccttg attagccgctacaagatcgt aaagagcgaa 6120 accgggcggc cggagtacat cgagatcgag ctagctgattggatgtaccg cgagatcaca 6180 gaaggcaaga acccggacgt gctgacggtt caccccgattactttttgat cgatcccggc 6240 atcggccgtt ttctctaccg cctggcacgc cgcgccgcaggcaaggcaga agccagatgg 6300 ttgttcaaga cgatctacga acgcagtggc agcgccggagagttcaagaa gttctgtttc 6360 accgtgcgca agctgatcgg gtcaaatgac ctgccggagtacgatttgaa ggaggaggcg 6420 gggcaggctg gcccgatcct agtcatgcgc taccgcaacctgatcgaggg cgaagcatcc 6480 gccggttcct aatgtacgga gcagatgcta gggcaaattgccctagcagg ggaaaaaggt 6540 cgaaaaggtc tctttcctgt ggatagcacg tacattgggaacccaaagcc gtacattggg 6600 aaccggaacc cgtacattgg gaacccaaag ccgtacattgggaaccggtc acacatgtaa 6660 gtgactgata taaaagagaa aaaaggcgat ttttccgcctaaaactcttt aaaacttatt 6720 aaaactctta aaacccgcct ggcctgtgca taactgtctggccagcgcac agccgaagag 6780 ctgcaaaaag cgcctaccct tcggtcgctg cgctccctacgccccgccgc ttcgcgtcgg 6840 cctatcgcgg ccgctggccg ctcaaaaatg gctggcctacggccaggcaa tctaccaggg 6900 cgcggacaag ccgcgccgtc gccactcgac cgccggcgctgaggtctgcc tcgtgaagaa 6960 ggtgttgctg actcatacca ggcctgaatc gccccatcatccagccagaa agtgagggag 7020 ccacggttga tgagagcttt gttgtaggtg gaccagttggtgattttgaa cttttgcttt 7080 gccacggaac ggtctgcgtt gtcgggaaga tgcgtgatctgatccttcaa ctcagcaaaa 7140 gttcgattta ttcaacaaag ccgccgtccc gtcaagtcagcgtaatgctc tgccagtgtt 7200 acaaccaatt aaccaattct gattagaaaa actcatcgagcatcaaatga aactgcaatt 7260 tattcatatc aggattatca ataccatatt tttgaaaaagccgtttctgt aatgaaggag 7320 aaaactcacc gaggcagttc cataggatgg caagatcctggtatcggtct gcgattccga 7380 ctcgtccaac atcaatacaa cctattaatt tcccctcgtcaaaaataagg ttatcaagtg 7440 agaaatcacc atgagtgacg actgaatccg gtgagaatggcaaaagctct gcattaatga 7500 atcggccaac gcgcggggag aggcggtttg cgtattgggcgctcttccgc ttcctcgctc 7560 actgactcgc tgcgctcggt cgttcggctg cggcgagcggtatcagctca ctcaaaggcg 7620 gtaatacggt tatccacaga atcaggggat aacgcaggaaagaacatgtg agcaaaaggc 7680 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctggcgtttttcca taggctccgc 7740 ccccctgacg agcatcacaa aaatcgacgc tcaagtcagaggtggcgaaa cccgacagga 7800 ctataaagat accaggcgtt tccccctgga agctccctcgtgcgctctcc tgttccgacc 7860 ctgccgctta ccggatacct gtccgccttt ctcccttcgggaagcgtggc gctttctcat 7920 agctcacgct gtaggtatct cagttcggtg taggtcgttcgctccaagct gggctgtgtg 7980 cacgaacccc ccgttcagcc cgaccgctgc gccttatccggtaactatcg tcttgagtcc 8040 aacccggtaa gacacgactt atcgccactg gcagcagccactggtaacag gattagcaga 8100 gcgaggtatg taggcggtgc tacagagttc ttgaagtggtggcctaacta cggctacact 8160 agaagaacag tatttggtat ctgcgctctg ctgaagccagttaccttcgg aaaaagagtt 8220 ggtagctctt gatccggcaa acaaaccacc gctggtagcggtggtttttt tgtttgcaag 8280 cagcagatta cgcgcagaaa aaaaggatct caagaagatcctttgatctt ttctacgggg 8340 tctgacgctc agtggaacga aaactcacgt taagggattttggtcatgag attatcaaaa 8400 aggatcttca cctagatcct tttgatccgg aattaattcctgtggttggc atgcacatac 8460 aaatggacga acggataaac cttttcacgc ccttttaaatatccgattat tctaataaac 8520 gctcttttct cttaggttta cccgccaata tatcctgtcaaacactgata gtttaaactg 8580 aaggcgggaa acgacaatct gatcatgagc ggagaattaagggagtcacg ttatgacccc 8640 cgccgatgac gcgggacaag ccgttttacg tttggaactgacagaaccgc aacgctgcag 8700 gaattggccg cagcggccat ttaaatcaat tgggcgcgccgaattcgagc tcggtac 8757

What is claimed is:
 1. A method for transforming soybean cells ortissue, comprising: (a) preparing an explant from a soybean seed by: (i)removing a hypocotyl from said soybean seed; (ii) removing one cotyledonalong with its adjacent axillary bud, leaving primary leaves attached toa remaining cotyledon; and (iii) removing a portion of a primary leaffrom said remaining cotyledon, thereby generating a primary leaf base;and (b) co-cultivating said explant with Agrobacterium comprising atleast one nucleic acid of interest to be incorporated into a genome ofone or more soybean cells.
 2. The method of claim 1, further comprisingcultivating at least one formed shoot in a medium containing a selectionagent.
 3. The method of claim 2, wherein said at least one nucleic acidof interest comprises a selectable marker gene.
 4. The method of claim3, wherein said selectable marker gene is a phosphomannose isomerasegene.
 5. The method of claim 4, wherein said selection agent is mannose.6. The method of claim 4, wherein co-cultivation with said Agrobacteriumis carried out in the presence of mannose.
 7. The method of claim 2,further comprising inducing shoot formation from said primary leaf base.8. The method of claim 7, wherein shoot formation is induced byculturing said primary leaf base in a medium comprising a shoot-inducinghormone.
 9. The method of claim 8, wherein said shoot-inducing hormonecomprises at least one of an auxin, a cytokinin, and a gibberellic acid.10. The method of claim 9, wherein said auxin is selected from the groupconsisting of IAA, NAA, and IBA.
 11. The method of claim 9, wherein saidcytokinin is selected from the group consisting of benzylaminopurine(BAP), thidiazuron, kinetin, and isopentenyl adenine.
 12. The method ofclaim 7, wherein induction of shoot formation comprises removing one ormore of a primary meristem, a secondary meristem, and an axillarymeristem attached to a cotyledon.
 13. The method of claim 7, furthercomprising selecting a transformed shoot.
 14. The method of claim 13,further comprising regenerating a selected transformed shoot into asoybean plant.
 15. The method of claim 1, wherein said soybean seed is amature seed.
 16. The method of claim 1, wherein said soybean seed is animmature seed.
 17. The method of claim 1, wherein said soybean seed is agerminated seed.
 18. A method for producing a stably transformed soybeanplant, comprising: (a) preparing an explant from a soybean seed by: (i)removing a hypocotyl from said soybean seed; (ii) removing one cotyledonalong with its adjacent axillary bud, leaving primary leaves attached toa remaining cotyledon; and (iii) removing a portion of each primary leaffrom said remaining cotyledon, thereby generating a pair of primary leafbases; (b) co-cultivating said explant with Agrobacterium comprising anucleic acid of interest to be incorporated into a genome of a soybeancell; (c) inducing shoot formation from each primary leaf base; (d)cultivating at least one formed shoot in a medium containing a selectionagent; (e) selecting a transformed shoot; and (f) regenerating aselected transformed shoot into a soybean plant.
 19. A transgenicsoybean plant regenerated from soybean cells or tissue transformedaccording to the method of claim
 1. 20. A transgenic seed produced bythe transgenic plant of claim
 19. 21. A transgenic soybean plantregenerated from soybean cells or tissue transformed according to themethod of claim
 18. 22. A transgenic seed produced by the transgenicplant of claim 21.